Liposome compositions comprising an adjuvant that activates or increases the activity of TLR2 and uses thereof

ABSTRACT

The invention provides compositions comprising liposomes, an antigen capable of inducing a humoral immune response, a carrier comprising a continuous phase of a hydrophobic substance, and an adjuvant that activates or increases the activity of TLR2. The invention also provides uses for such compositions in inducing a humoral response and methods for their use in the treatment of a disease, disorder or ailment ameliorated by a humoral immune response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 61/544,020 filed on Oct. 6, 2011, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present application relates to vaccine compositions that enhance theproduction of antigen-specific antibodies in immunized subjects.

BACKGROUND OF THE INVENTION

Immune responses induced by vaccination can be categorized broadly intohumoral or cellular types. A humoral response is typically desired toprotect against viral or bacterial invaders, whereas immunity againstvirally infected cells and cancer cells typically involves a cellmediated response. Humoral immunity is typified by high levels ofantibody production by B cells, whereas cellular immunity ischaracterized by increased activation of cytotoxic CD8 T lymphocytes.

The type of immunity induced by a vaccine largely depends on the type ofadjuvant included in the vaccine. Adjuvants based on palmitic acid, suchas dipalmitoyl-S-glyceryl-cysteine (PAM₂Cys) andtripalmitoyl-S-glyceryl-cysteine (PAM₃Cys) and variants thereof, havebeen reported to enhance humoral and cellular responses against avariety of antigens. For practical reasons, the solubility of suchadjuvants has been typically improved with the addition of hydrophilicnon-immunogenic amino acid residues (Lysines for example). Suchadjuvants have been mixed with antigen but in many instances palmiticacid based adjuvants have been covalently linked to antigens beforeadministering to a subject. Palmitic acid adjuvants have also beenco-delivered with antigen using liposomes as carriers. Protein based andcarbohydrate based antigens have been combined with palmitic acidadjuvants to produce antibody and T cell responses. The use of palmiticacid adjuvants for cancer applications is well documented, with activitymediated primarily by cellular responses.

Although palmitic acid derivatives are known to proliferate B cells,induce isotypic switching, induce differentiation of human B lymphocytesto IgG secreting plasma cells and increase expression of severalco-stimulatory molecules (MHC I, II, CD80, etc), reports of palmiticacid adjuvants inducing antibody responses has varied in the literaturefrom being able to enhance antibody responses to not being particularlyuseful for generating such responses.

Thus, there remains a need for the development of vaccine compositionsfor generating strong humoral responses against a variety of antigens.The present invention provides vaccine compositions that contain alipid-based adjuvant and are particularly useful for inducing a highlevel of antibodies in immunized subjects.

SUMMARY OF THE INVENTION

In one aspect, there is provided a composition comprising: liposomes; anantigen capable of inducing a humoral immune response; a carriercomprising a continuous phase of a hydrophobic substance; and anadjuvant which activates or increases the activity of the toll-likereceptor 2 (TLR2), for example, by interacting with a TLR2 dimer such asTLR1/2 or TLR2/6.

In an embodiment of the composition as described herein, the adjuvant isa lipid-based adjuvant.

In an embodiment of the composition as described herein, the lipid-basedadjuvant is a palmitic acid adjuvant.

In an embodiment of the composition as described herein, the lipid-basedadjuvant comprises dipalmitoyl-S-glyceryl-cysteine (PAM₂Cys) ortripalmitoyl-S-glyceryl-cysteine (PAM₃Cys); or the lipid-based adjuvantis Pam-2-Cys-Ser-(Lys)4 (SEQ ID NO: 1) or Pam-3-Cys-Ser-(Lys)4 (SEQ IDNO: 1).

In another embodiment of the composition as described herein, thelipid-based adjuvant is, or comprises: the synthetic diacylatedlipoprotein FSL-1 (Pam2CGDPKHPKSF) (SEQ ID NO: 2), a syntheticlipoprotein derived from Mycoplasma salivarium, or themacrophage-activating lipopeptide (MALP-2) from Mycoplasma fermentans.

In an embodiment, the composition of the invention may comprise anadjuvant as described herein in combination with at least one othersuitable adjuvant.

In another embodiment of the composition as described herein, theantigen is a polypeptide or a carbohydrate.

In an embodiment of the composition as described herein, the antigencomprises a B cell epitope, or a plurality of B cell epitopes.

In another embodiment of the composition as described herein, theantigen is a membrane surface-bound cancer antigen; a toxin; an allergensuch as pollen; or an amyloid protein.

In another embodiment of the composition as described herein, theliposome comprises a phospholipid or unesterified cholesterol.

In another embodiment, the composition as described herein is capable ofinducing a humoral immune response in a subject with a single dose.

The present invention in a further aspect provides a method for treatingor preventing a disease or disorder ameliorated by a humoral immuneresponse, said method comprising administering the composition asdescribed herein to a subject.

In another aspect, the present invention provides a method for treatingor preventing an infectious disease; a cancer involving a membranesurface-bound cancer antigen; or a disease or disorder where it isdesirable to sequester antigen in circulation, such as an amyloidprotein for treating e.g. Alzheimer's disease, said method comprisingadministering the composition as described herein to a subject.

In another aspect, the present invention provides a method forneutralizing a toxin, virus, bacterium or allergen, with an antibody,said method comprising administering the composition as described hereinto a subject.

In an embodiment of the invention, the subject referred to herein is amammal.

In another embodiment of the invention, the subject referred to hereinis a human.

According to another aspect, the present invention relates to a kituseful for treating or preventing a disease or disorder as describedherein, or neutralizing a toxin, virus, bacterium or allergen, with anantibody, wherein the kit comprises a composition as described herein,and instructions for its use thereof.

According to another aspect, the present invention relates to a methodof making a composition of the present invention as described herein.

Other aspects and features of the present invention will become apparentto those of ordinary skill in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

In the figures, which illustrate embodiments of the invention by way ofexample only:

FIG. 1 illustrates the humoral immune response generated by a vaccinemade in accordance with the invention (“Vaccine A”). Two groups of mice(n=8 or 9) were vaccinated as follows: Group 1 mice were vaccinated witha single dose of 1 microgram rHA and 1 microgram Pam-3-Cys-Ser-(Lys)4(SEQ ID NO: 1) in a 50 microliter dose formulated as awater-free/liposome/P3C/hydrophobic carrier vaccine (Vaccine A). Group 2mice were treated with 1 microgram rHA and 50 micrograms alum per 50microliter dose of control alum vaccine; mice were boosted 28 dayspost-vaccination. Humoral immune responses were measured by ELISA asdescribed above. For each treatment group, the log 10 values of theendpoint antibody titers were averaged and standard deviationscalculated for each time point.

FIG. 2 illustrates the effect of a single administration of a vaccinemade in accordance with the invention (“Vaccine B”). Three groups ofmice (n=9 or 11) were vaccinated as follows: Group 1 mice werevaccinated with a single dose of 1 microgram PT and 1 microgramPam-3-Cys-Ser-(Lys)4 (SEQ ID NO: 1) in a 50 microliter dose formulatedas a water-free/liposome/P3C/hydrophobic carrier vaccine (Vaccine B).Group 2 and group 3 mice were treated with 1 microgram PT and 100micrograms alum per 100 microliter dose of control alum vaccine; group 2mice received a single dose, group 3 mice were boosted at days 21 and31. Group 4 mice remained un-vaccinated. Mice were challenged 56 dayspost-vaccination with aerosol inoculation with Bordetella pertussis andbacterial lung counts established 8 and 15 days post-challenge. For eachtreatment group, the log 10 values of the colony forming units per lungwere averaged and standard deviations calculated for each time point.

FIG. 3 illustrates the effect of a single administration of a vaccinemade in accordance with the invention (“Vaccine C”). Two groups ofrabbits (N=8) were vaccinated as follows: Group 1 rabbits werevaccinated with a single dose of 8 micrograms rPA and 2 microgramsPam-3-Cys in a 100 microliter dose formulated as aliposome/P3C/hydrophobic carrier vaccine (Vaccine C). Group 2 rabbitswere treated with 8 micrograms rPA and 350 micrograms aluminum hydroxideper 100 microliter dose of control alum vaccine; rabbits were boosted at28 and 84 days post-vaccination. Humoral immune responses were measuredby ELISA as described above. For each treatment group, the log 10 valuesof the endpoint antibody titers were averaged and standard deviationscalculated for each time point.

FIG. 4 illustrates that Vaccine A of the invention, specificallycomprising an antigen, liposomes, a palmitic acid adjuvant and ahydrophobic carrier, is capable of simulating maximal immunogenicity.Four groups of mice (N=10) were vaccinated as follows: Group 1 mice werevaccinated with a single dose of 1 microgram rHA and 1 microgramPam-3-Cys in a 50 microliter dose formulated as aliposome/P3C/hydrophobic carrier vaccine (Vaccine A, the invention).Group 2 mice were treated with 1 microgram of rHA and 1 microgram of P3Cper 50 microliter dose. Group 3 mice were treated with 1 microgram rHAand 1 microgram of P3C per 50 microliter dose formulated as anaqueous/liposome/P3C vaccine. Group 4 mice were treated with 1 microgramof rHA formulated as a liposome/hydrophobic carrier vaccine. Humoralresponses were measured by ELISA as described above. For each treatmentgroup, the log 10 values of the endpoint antibody titers were averagedand standard deviations calculated for each time point.

FIG. 5 illustrates that Vaccine D of the invention, comprising alipid-based adjuvant (i.e. Pam-3-Cys), is capable of enhancing theimmune response to inactivated viral vaccine formulations. Panel (A)shows the clinical score and Panel (B) shows the overall survival ofmice challenged with A/PR/8/34 (H1N1) influenza 28 days after a singlevaccination. Group 1 mice were treated with 50 microliters of saline.Group 2 mice were treated with 50 microliters of 2.56×10^3 TCID50A/PR/8/34 with alum. Group 3 mice were treated with 50 microliters of2.56×10^3 TCID50 A/PR/8/34 and 1 microgram Pam-3-Cys formulated as aliposome/P3C/hydrophobic carrier vaccine (Vaccine D). Following viralchallenge, clinical signs for each mouse were followed every day for 10days, and scored on the basis of physical appearance, posture, activitylevel/behavior, body temperature, body weight and hydration. Any mousewith a score of >12 (out of a total possible 18) was euthanized.

FIG. 6 illustrates that Vaccine A of the invention is capable ofstimulating a specific immune response which is significantly strongerthan a comparable vaccine prepared with a different adjuvant(liposomes/IMQ/hydrophobic carrier). Two groups of mice (N=9) werevaccinated as follows: Group 1 mice were vaccinated with a single doseof 1 microgram rHA and 1 microgram Pam-3-Cys in a 50 microliter doseformulated as a liposome/P3C/hydrophobic carrier vaccine (Vaccine A).Group 2 mice were treated with 1 microgram of rHA and 1 microgram ofImiquimod per 50 microliter dose formulated as aliposome/IMQ/hydrophobic carrier vaccine (Control Vaccine). Humoralimmune responses were measured by ELISA as described above. For eachtreatment group, the log 10 values of the endpoint antibody titers werecalculated. Statistical analysis performed by unpaired t-test, P<0.005.

FIG. 7 illustrates that both Pam3Cys and Pam2Cys are capable of inducingpotent proliferation of B cells. Purified B cells from C57BL6 mice werestimulated for three days in vitro with Pam2Cys (A), Pam3Cys (B). PolyI:C (C) or LPS (D) at three different concentrations in the presence ofanti-Ig & anti-CD40. Proliferation was measured by [³H]-thymidineincorporation, quantified as counts per minute (CPM). N=2-10,statistical analysis performed by ANOVA.

DETAILED DESCRIPTION

The type of immunity induced by a vaccine largely depends on the type ofadjuvant included in the vaccine. The magnitude and duration of such aresponse depends on the type of adjuvant used as well as the compositionor method by which the antigen and adjuvant are presented to the immunesystem. For example, live attenuated viruses can be used to delivergenes for antigens of interest that are then produced in vivo to bereadily presented by antigen presenting cells; liposomes can be used toco-deliver antigen and adjuvant directly to antigen presenting cells todrive a better immune response than naked antigen and adjuvant arecapable of.

The present invention provides vaccine compositions that use an adjuvantthat activates or increases the activity of TLR2 to generateunexpectedly strong antibody responses. In some embodiments, vaccinecompositions of the invention were capable of protecting a subject froma disease agent with as little as a single dose, whereas, as shown inthe examples herein, control vaccines were unable to produce suchantibody levels and are incapable of protecting vaccinated subjects tothe same degree.

Compositions of the invention, combining an antigen capable of inducinga humoral immune response, an adjuvant that activates or increases theactivity of TLR2, liposomes and a carrier comprising a continuous phaseof a hydrophobic substance provided surprisingly higher antibody titersthan aqueous aluminum based control compositions. Furthermore, a singledose of a composition of the invention was able to effectively protectmice from bacterial challenge and allow them to completely clear theinfection from the lungs, which was not observed for aqueous aluminumbased control compositions.

The ability to raise robust and long lasting humoral immune responseswith at least one immunization using the components of the describedcomposition of the invention (e.g. Examples 1 to 4) illustrates theparticular usefulness of these compositions in a wide range of medicalapplications, such as for example those described herein. As shown inthe examples herein, compositions of the invention can produce a strongand enhanced immune response at least as early as three weekspost-immunization, and the immune response is long-lived with antibodytiters remaining high for at least twenty-four weeks post-immunization(e.g. FIG. 3).

Compositions of the invention, comprising antigen, liposomes, anadjuvant that activates or increases the activity of TLR2, and a carriercomprising a continuous phase of a hydrophobic substance, may raiserobust and long lasting humoral immune responses. For example, the datadescribed in Example 4 herein shows that antibody titers generated bymice in Group 1 (a vaccine of the invention) were significantly higherthan the antibody titers generated by mice in control groups withoutliposomes (Group 2), without hydrophobic carrier (Group 3), or withoutlipid-based adjuvant (Group 4).

Thus, vaccine compositions of the invention containing a lipid-basedadjuvant are capable of generating strong humoral immune responses, withhigh levels of antibody production, in immunized subjects.

Compositions as described herein may be useful for treating orpreventing diseases and/or disorders ameliorated by humoral immuneresponses (e.g. involving B-cells and antibody production). Thecompositions find application in any instance in which it is desired toadminister an antigen to a subject to induce a humoral immune responseor antibody production.

As used herein, to “induce” an immune response is to elicit and/orpotentiate an immune response. Inducing an immune response encompassesinstances where the immune response is enhanced, elevated, improved orstrengthened to the benefit of the host relative to the prior immuneresponse status, for example, before the administration of a compositionof the invention.

A humoral immune response, as opposed to cell-mediated immunity, ismediated by secreted antibodies which are produced in the cells of the Blymphocyte lineage (B cells). Such secreted antibodies bind to antigens,such as for example those on the surfaces of foreign substances and/orpathogens (e.g. viruses, bacteria, etc.) and flag them for destruction.

An “antibody” is a protein comprising one or more polypeptidessubstantially or partially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include theκ, λ, α, γ, δ, ε and μ constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither K or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (antibody) structural unitcomprises a protein containing four polypeptides. Each antibodystructural unit is composed of two identical pairs of polypeptidechains, each having one “light” and one “heavy” chain. The N-terminus ofeach chain defines a variable region primarily responsible for antigenrecognition. Antibody structural units (e.g. of the IgA and IgM classes)may also assemble into oligomeric forms with each other and additionalpolypeptide chains, for example as IgM pentamers in association with theJ-chain polypeptide.

Antibodies are the antigen-specific glycoprotein products of a subset ofwhite blood cells called B lymphocytes (B cells). Engagement of antigenwith antibody expressed on the surface of B cells can induce an antibodyresponse comprising stimulation of B cells to become activated, toundergo mitosis and to terminally differentiate into plasma cells, whichare specialized for synthesis and secretion of antigen-specificantibody.

B cells are the sole producers of antibodies during an immune responseand are thus a key element to effective humoral immunity. In addition toproducing large amounts of antibodies, B cells also act asantigen-presenting cells and can present antigen to T cells, such as Thelper CD4 or cytotoxic CD8, thus propagating the immune response. Bcells, as well as T cells, are part of the adaptive immune responsewhich is essential for vaccine efficacy. During an active immuneresponse, induced either by vaccination or natural infection,antigen-specific B cells are activated and clonally expand. Duringexpansion, B cells evolve to have higher affinity for the epitope.Proliferation of B cells can be induced indirectly by activated T-helpercells, and also directly through stimulation of receptors, such as thetoll-like receptors (TLRs).

Antigen presenting cells, such as dendritic cells and B cells, are drawnto vaccination sites and can interact with antigens and adjuvantscontained in the vaccine. The adjuvant stimulates the cells to becomeactivated and the antigen provides the blueprint for the target.Different types of adjuvants provide different stimulation signals tocells. For example, Poly I:C (a TLR3 agonist) can activate dendriticcells, but not B cells. Adjuvants such as Pam3Cys, Pam2Cys and FSL-1 areespecially adept at activating and initiating proliferation of B cells,which is expected to facilitate the production of an antibody response(Moyle et al., Curr Med Chem, 2008; So, J Immunol, 2012).

As used herein, the term “antibody response” refers to an increase inthe amount of antigen-specific antibodies in the body of a subject inresponse to introduction of the antigen into the body of the subject.

One method of evaluating an antibody response is to measure the titersof antibodies reactive with a particular antigen. This may be performedusing a variety of methods known in the art such as enzyme-linkedimmunosorbent assay (ELISA) of antibody-containing substances obtainedfrom animals. For example, the titers of serum antibodies which bind toa particular antigen may be determined in a subject both before andafter exposure to the antigen. A statistically significant increase inthe titer of antigen-specific antibodies following exposure to theantigen would indicate the subject had mounted an antibody response tothe antigen.

Other assays that may be used to detect the presence of anantigen-specific antibody include, without limitation, immunologicalassays (e.g. radioimmunoassay (RIA)), immunoprecipitation assays, andprotein blot (e.g. Western blot) assays; and neutralization assays(e.g., neutralization of viral infectivity in an in vitro or in vivoassay).

The compositions of the present invention, by stimulating strongantibody responses, may be capable of protecting a subject from adisease, disorder or ailment associated with an antigen capable ofinducing a humoral immune response.

Without limitation, this includes for example, infectious diseases,cancers involving a membrane surface-bound cancer antigen which isrecognized by an antibody, diseases where it is desirable to sequesterantigen in circulation, like amyloid protein (e.g. Alzheimer's disease);neutralizing toxins with an antibody; neutralizing viruses or bacteriawith an antibody; or neutralizing allergens (e.g. pollen) for thetreatment of allergies.

“Humoral immune response” as referred to herein relates to antibodyproduction and the accessory processes that accompany it, such as forexample T-helper 2 (Th2) cell activation and cytokine production,isotype switching, affinity maturation and memory cell activation. Italso refers to the effector functions of an antibody, such as forexample toxin neutralization, classical complement activation, andpromotion of phagocytosis and pathogen elimination. The humoral immuneresponse is aided by CD4+ Th2 cells and therefore the activation orgeneration of this cell type is also indicative of a humoral immuneresponse as referred to herein.

A “humoral immune response” as referred to herein may also encompass thegeneration and/or activation of T-helper 17 (Th17) cells. Th17 cells area subset of helper-effector T-lymphocytes characterized by the secretionof host defense cytokines such as IL-17, IL-17F and IL-22. Th17 cellsare considered developmentally distinct from Th1 and Th2 cells, and havebeen postulated to facilitate the humoral immune response, such as forexample, providing an important function in anti-microbial immunity andprotecting against infections. Their production of IL-22 is thought tostimulate epithelial cells to produce anti-microbial proteins andproduction of IL-17 may be involved in the recruitment, activation andmigration of neutrophils to protect against host infection by variousbacterial and fungal species.

A humoral immune response is the main mechanism for effective infectiousdisease vaccines. However, a humoral immune response can also be usefulfor combating cancer. Unlike a cancer vaccine designed to produce acytotoxic CD8 T cell response that can recognize and destroy cancercells, B cell mediated responses may target cancer cells through othermechanisms which may in some instances cooperate with a cytotoxic CD8 Tcell for maximum benefit. Examples of mechanisms of B cell mediated(e.g. humoral immune response mediated) anti-tumor responses include,without limitation: 1) Antibodies produced by B cells that bind tosurface antigens found on tumor cells or other cells that influencetumorigenesis. Such antibodies can, for example, induce killing oftarget cells through antibody-dependant cell-mediated cytotoxicity(ADCC) or complement fixation, potentially resulting in the release ofadditional antigens that can be recognized by the immune system; 2)Antibodies that bind to receptors on tumor cells to block theirstimulation and in effect neutralize their effects; 3) Antibodies thatbind to factors released by or associated with tumor or tumor-associatedcells to modulate a signaling or cellular pathway that supports cancer;and 4) Antibodies that bind to intracellular targets and mediateanti-tumor activity through a currently unknown mechanism.

Several methods can be used to demonstrate the induction of humoralimmunity following vaccination. These can be broadly classified intodetection of: i) specific antigen presenting cells; ii) specificeffector cells and their functions; and iii) release of solublemediators such as cytokines.

i) Antigen Presenting Cells:

Dendritic cells and B-cells (and to a lesser extent macrophages) areequipped with special immuno-stimulatory receptors that allow forenhanced activation of T cells, and are termed professional antigenpresenting cells (APC). These immuno-stimulatory molecules (also calledas co-stimulatory molecules) are up-regulated on these cells followinginfection or vaccination, during the process of antigen presentation toeffector cells such as CD4 and CD8 cytotoxic T cells. Suchco-stimulatory molecules (such as CD80, CD86, MHC class I or MHC classII) can be detected by using flow cytometry with fluorochrome-conjugatedantibodies directed against these molecules along with antibodies thatspecifically identify APC (such as CD11c for dendritic cells).

ii) CD4+“Helper” T-Cells:

CD4+ lymphocytes, or helper T cells, are immune response mediators, andplay an important role in establishing and maximizing the capabilitiesof the adaptive immune response. These cells have no cytotoxic orphagocytic activity; and cannot kill infected cells or clear pathogens,but, in essence “manage” the immune response, by directing other cellsto perform these tasks. Two types of effector CD4+ T helper cellresponses can be induced by a professional APC, designated Th1 and Th2,each designed to eliminate different types of pathogens.

Helper T cells express T-cell receptors (TCR) that recognize antigenbound to Class II MHC molecules. The activation of a naive helper T-cellcauses it to release cytokines, which influences the activity of manycell types, including the APC that activated it. The two Th cellpopulations, Th1 and Th2, differ in the pattern of the effector proteins(cytokines) produced. In general, Th1 cells assist the cellular immuneresponse by activation of macrophages and cytotoxic T-cells; whereas Th2cells promote the humoral immune response by stimulation of B-cells forconversion into plasma cells and by formation of antibodies. A responseregulated by Th2 type cells may predominantly enhance the production ofIgG1 in mouse (IgG2 in humans). The measure of cytokines associated withTh1 or Th2 responses will give a measure of successful vaccination. Thiscan be achieved by specific ELISA designed for Th1-cytokines such asIFN-γ, IL-2, IL-12, TNF-α and others, or Th2-cytokines such as IL-4,IL-5, IL10 among others.

Another Th cell population is the Th17 cell. The measure of cytokinesassociated with Th17 cells can also give a measure of a successfulvaccination. This can be achieved, for example, by specific ELISAdesigned for Th17 cytokines such as IL-17, IL-17F and IL-22.

iii) Measurement of cytokines:

released from regional lymph nodes gives a good indication of successfulimmunization. As a result of antigen presentation and maturation of APCand immune effector cells such as CD4 and CD8 T cells, several cytokinesare released by lymph node cells. By culturing these LNC in vitro in thepresence of antigen, an antigen-specific immune response can be detectedby measuring release if certain important cytokines such as IL-4, IL-5,and IL10 for detection of a humoral immune response. This could be doneby ELISA using culture supernatants and recombinant cytokines asstandards.

Successful immunization may further be determined in a number ofadditional ways known to the skilled person including, but not limitedto, hemagglutination inhibition (HAI) and serum neutralizationinhibition assays to detect functional antibodies; challenge studies, inwhich vaccinated subjects are challenged with the associated pathogen todetermine the efficacy of the vaccination; and the use of fluorescenceactivated cell sorting (FACS) to determine the population of cells thatexpress a specific cell surface marker, e.g. in the identification ofactivated or memory lymphocytes. Also, vaccine efficacy in stimulating ahumoral immune response can be assessed by ELISA detection ofantigen-specific antibody levels in the serum of immunized subjects. Askilled person may also determine if immunization with a composition ofthe invention elicited a humoral (or antibody mediated) response usingother known methods. See, for example, Current Protocols in ImmunologyColigan et al., ed. (Wiley Interscience, 2007). The term “infectiousdisease”, as used herein, may refer for example to any communicabledisease, contagious disease or transmissible disease resulting from theinfection, presence and/or growth of pathogenic biological agents.Without limitation, an infectious pathogenic agent may include forexample viruses, bacteria, fungi, protozoa, and parasites. Non-limitingexamples of infectious diseases include influenza (e.g. infection byinfluenza virus), respiratory tract infections such as, for example,bronchiolitis and pneumonia (e.g. infection by respiratory syncytialvirus), pertussis or whooping cough (e.g. infection by Bordetellapertussis), anthrax (e.g. infection by Bacillus anthracis) and malaria(e.g. infection by Plasmodium malariae, Plasmodium falciparum,Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi).

As used herein, the terms “cancer”, “cancer cells”, “tumor” and “tumorcells”, (used interchangeably) refer to cells that exhibit abnormalgrowth, characterized by a significant loss of control of cellproliferation or cells that have been immortalized. The term “cancer” or“tumor” includes metastatic as well as non-metastatic cancer or tumors.A cancer may be diagnosed using criteria generally accepted in the art,including the presence of a malignant tumor.

A “toxin”, as used herein, refers to any substance produced by livingcells or organisms (e.g. plants, animals, microorganisms, etc.) that iscapable of causing a disease or ailment, or an infectious substance, ora recombinant or synthesized molecule capable of adverse effect. Toxinsmay be for example small molecules, peptides, or proteins. Toxinsinclude drug substances such as, for example, cocaine.

An “allergen”, as used herein, refers to any substance that can cause anallergy. The allergen and may be derived from, without limitation,cells, cell extracts, proteins, polypeptides, peptides, polysaccharides,polysaccharide conjugates, peptide and non-peptide mimics ofpolysaccharides and other molecules, small molecules, lipids,glycolipids, and carbohydrates of plants, animals, fungi, insects, food,drugs, dust, and mites. Allergens include but are not limited toenvironmental aeroallergens; plant pollens (e.g. ragweed/hayfever); weedpollen allergens; grass pollen allergens; Johnson grass; tree pollenallergens; ryegrass; arachnid allergens (e.g. house dust miteallergens); storage mite allergens; Japanese cedar pollen/hay fever,mold/fungal spore allergens; animal allergens (e.g., dog, guinea pig,hamster, gerbil, rat, mouse, etc., allergens); food allergens (e.g.crustaceans; nuts; citrus fruits; flour; coffee); insect allergens (e.g.fleas, cockroach); venoms: (Hymenoptera, yellow jacket, honey bee, wasp,hornet, fire ant); bacterial allergens (e.g. streptococcal antigens;parasite allergens such as Ascaris antigen); viral antigens; drugallergens (e.g. penicillin); hormones (e.g. insulin); enzymes (e.g.streptokinase); and drugs or chemicals capable of acting as incompleteantigens or haptens (e.g. the acid anhydrides and the isocyanates).

Where a hapten is used in a composition of the invention, it may beattached to a carrier, such as for example a protein, to form ahapten-carrier adduct. The hapten-carrier adduct is capable ofinitiating a humoral immune response, whereas the hapten itself wouldnot elicit antibody production. Non-limiting examples of haptens areaniline, urushiol (a toxin in poison ivy), hydralazine, fluorescein,biotin, digoxigenin and dinitrophenol.

“Treating” or “treatment of”, or “preventing” or “prevention of”, asreferred to herein refers to an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilisation of the state of disease, prevention ofdevelopment of disease, prevention of spread of disease, delay orslowing of disease progression, delay or slowing of disease onset,conferring protective immunity against a disease-causing agent andamelioration or palliation of the disease state. “Treating” or“preventing” can also mean prolonging survival of a patient beyond thatexpected in the absence of treatment and can also mean inhibiting theprogression of disease temporarily, although more preferably, itinvolves preventing the occurrence of disease such as by preventinginfection in a subject.

The subject to be treated may be any vertebrate, preferably a mammal,more preferably a human.

Adjuvants

Suitable adjuvants of the composition of the invention are adjuvantsthat activate or increases the activity of TLR2. In some embodiments,the adjuvant is a lipid-based adjuvant, which encompasses any adjuvantthat comprises at least one lipid moiety or lipid component.

As used herein, the expression “lipid moiety” or “lipid component”refers to any fatty acid (e.g. fatty acyls) or derivative thereof,including for example triglycerides, diglycerides, and monoglycerides.Exemplary fatty acids include, without limitation, palmitoyl, myristoyl,stearoyl and decanoyl groups or any C2 to C30 saturated or unsaturatedfatty acyl group, preferably any C14 to C22 saturated or unsaturatedfatty acyl group, and more preferably a C16 saturated or unsaturatedfatty acyl group. Thus, as referred to herein, the expression“lipid-based adjuvant” encompasses any adjuvant comprising a fatty acylgroup or derivative thereof.

Lipid-based adjuvants of the present invention contain at a minimum atleast one lipid moiety, or a synthetic/semi-synthetic lipid moietyanalogue, which can be coupled onto an amino acid, an oligopeptide orother molecules (e.g. a carbohydrate, a glycan, a polysaccharide,biotin, Rhodamine, etc.). Thus, without limitation, the lipid-basedadjuvant may be, for example, a lipoamino acid, a lipopeptide, alipoglycan, a lipopolysaccharide or a lipoteichoic acid. Moreover, alipid moiety or a structure containing a lipid moiety can be coupledcovalently or non-covalently to an antigen to create antigenic compoundswith built-in adjuvanting properties. For example, and withoutlimitation, the lipid-based moiety may comprise a cation (e.g. nickel)to provide a positive charge for non-covalent coupling.

In some embodiments, the lipid moiety or lipid component may benaturally occurring, such as for example a cell-wall component (e.g.lipoprotein) from a Gram-positive or Gram-negative bacteria,Rhodopseudomonas viridis, or mycoplasma. In other embodiments, the lipidmoiety or lipid component may be synthetic or semi-synthetic.

The lipid-based adjuvant may comprise palmitic acid (PAM) as at leastone of the lipid moieties or components of the adjuvant. Suchlipid-based adjuvants are referred to herein as a “palmitic acidadjuvant”. Palmitic acid is a low molecular weight lipid found in theimmunologically reactive Braun's lipoprotein of Escherichia coli. Othercommon chemical names for palmitic acid include, for example,hexadecanoic acid in IUPAC nomenclature and 1-Pentadecanecarboxylicacid. The molecular formula of palmitic acid is CH₃(CH₂)₁₄CO₂H. As willbe understood to those skilled in the art, it is possible that the lipidchain of palmitic acid may be altered. Exemplary compounds which may beused herein as palmitic acid adjuvants, and methods for their synthesis,are described for example in United States Patent Publications US2008/0233143; US 2010/0129385; and US 2011/0200632, the disclosures ofwhich are incorporated herein.

As described above for lipid moieties generally, a palmitic acidadjuvant contains at a minimum at least one palmitic acid moiety, whichcan be coupled onto an amino acid, an oligopeptide or other molecules. Apalmitic acid moiety or a structure containing palmitic acid can becoupled covalently or non-covalently to an antigen to create antigeniccompounds with built-in adjuvanting properties. The palmitic acid moietyor a chemical structure containing palmitic acid can be conjugated to acysteine peptide (Cys) to allow for various structural configurations ofthe adjuvant, including linear and branched structures. The cysteineresidue has been commonly extended by polar residues such as Serine(Ser) and/or lysine (Lys) at the C terminus to create adjuvant compoundswith improved solubility. Palmitic acid containing adjuvant compoundscould be admixed with an antigen, associated with antigen throughnon-covalent interactions, or alternatively covalently linked to anantigen, either directly or with the use of a linker/spacer, to generateenhanced immune responses. Most commonly, two palmitic acid moieties areattached to a glyceryl backbone and a cysteine residue to createdipalmitoyl-S-glyceryl-cysteine (PAM₂Cys) ortripalmitoyl-S-glyceryl-cysteine (PAM₃Cys), which can also be used inmultiple configurations as described above.

Palmitic acid adjuvants are known to activate B cells causing rapidproliferation and production of antibodies. B cells recognize theantigen co-delivered with the adjuvant in the vaccine formulation andthrough affinity maturation will proliferate with increasing specificitytowards the antigen. Activated 8 cells are known to secrete largequantities of soluble immunoglobin antibodies that can bind to solubletargets, such as bacteria, present in the blood. Antibody effectorfunctions are i) opsonization; ii) antibody-dependent cell-mediatedcytotoxicity (ADCC); iii) complement activation; iv) neutralization.While the majority of the B cells will mature into antibody secretingplasma cells, a portion should differentiate into memory B cells thatpersist after the immune response has controlled infection. Thisprovides long-term immunity against subsequent exposure to the pathogen.Ideally, a prophylactic vaccine should induce a strong memory B cellpopulation.

Therefore, in an embodiment, the adjuvant of the composition of theinvention is any type of adjuvant comprising a palmitic acid moiety orcomponent. The palmitic acid moiety may be modified or manipulated toimprove its stability in vitro or in vivo, enhance its binding toreceptors (such as for example toll-like receptors as described below)or enhance its biological activity.

In a particular embodiment, the palmitic acid adjuvant may comprisePAM₂Cys.

In another particular embodiment, the palmitic acid adjuvant maycomprise PAM₃Cys.

In another particular embodiment, the palmitic acid adjuvant may bePam-2-Cys-Ser-(Lys)4 (SEQ ID NO: 1) or Pam-3-Cys-Ser-(Lys)4 (SEQ ID NO:1). Such palmitic acid adjuvants are available, for example, as researchreagents from EMC Microcollections GmbH (Germany) and InvivoGen (SanDiego, Calif., USA).

Also available from EMC Microcollections are various analogs ofPam-2-Cys-Ser-(Lys)4 (SEQ ID NO: 1) and Pam-3-Cys-Ser-(Lys)4 (SEQ ID NO:1), including labelled analogs. These analogs are encompassed herein andinclude, without limitation, PAM₃Cys-SKKKK (SEQ ID NO: 1)(β-irradiated), R-PAM₃Cys-SKKKK (SEQ ID NO: 1), S-PAM₃Cys-SKKKK (SEQ IDNO: 1), PAM₃Cys-SKKKK(Biotin-Aca-Aca) (SEQ ID NO: 1),PAM₃Cys-SKKKK(Fluorescein-Aca-Aca) (SEQ ID NO: 1),PAM₃Cys-SKKKK(Rhodamine-Aca-Aca) (SEQ ID NO: 1), PAM₃Cys-SKKKK-FLAG-tag(SEQ ID NO: 1), PHC-SKKKK (SEQ ID NO: 3), PHC-SKKKK(Biotin-Aca-Aca) (SEQID NO: 3), PAM₃Cys-SSNAKIDQLSSDVQT (SEQ ID NO: 4),PAM₃Cys-SSNKSTTGSGETTTA (SEQ ID NO: 5), PAM₃Cys-SSTKPVSQDTSPKPA (SEQ IDNO: 6), PAM₃Cys-SSGSKPSGGPLPDAK (SEQ ID NO: 7), PAM₃Cys-SSGNKSAPSSSASSS(SEQ ID NO: 8), PAM₃Cys-GSHQMKSEGHANMQL (SEQ ID NO: 9),PAM₃Cys-SSSNNDAAGNGAAQT (SEQ ID NO: 10), PAM₃Cys-KQNVSSLDEKNSVSV (SEQ IDNO: 11), PAM₃Cys-NNSGKDGNTSANSAD (SEQ ID NO: 12),PAM₃Cys-NNGGPELKSDEVAKS (SEQ ID NO: 13), PAM₃Cys-SQEPAAPAAEATPAG (SEQ IDNO: 14), PAM₃Cys-SSSKSSDSSAPKAYG (SEQ ID NO: 15),PAM₃Cys-AQEKEAKSELDYDQT (SEQ ID NO: 16), Pam₂Cys-SKKKK (mixture of RRand RS stereoisomers) (SEQ ID NO: 1), R-Pam₂Cys-SKKKK (RR stereoisomer)(SEQ ID NO: 1), S-Pam₂Cys-SKKKK (RS stereoisomer) (SEQ ID NO: 1),Pam₂Cys(Pam)-SKKKK (SEQ ID NO: 3), Pam₂Cys-SKKKK(Biotin-Aca-Aca)-NH₂(SEQ ID NO: 1), Pam₂Cys-SKKKK(Fluorescein-Aca-Aca)-NH₂ (SEQ ID NO: 1),PAM₂Cys-SKKKK(Rhodamine-Aca-Aca)

—NH₂ (SEQ ID NO: 1), and PAM₂Cys-SKKKK-FLAG-tag (SEQ ID NO: 1). Whereappropriate, the palmitic acid adjuvant or analog thereof may used asstereochemically defined compounds or as a mixture of stereoisomers.

The adjuvant is one that activates or increases the activity oftoll-like receptors (TLRs), and preferably activates or increases theactivity of TLR2. As used herein, an adjuvant which “activates” or“increases the activity” of a TLR includes any adjuvant, in someembodiments a lipid-based adjuvant, which acts as a TLR agonist.Further, activating or increasing the activity of TLR2 encompasses itsactivation in any monomeric, homodimeric or heterodimeric form, andparticularly includes the activation of TLR2 as a heterodimer with TLR1or TLR6 (i.e. TLR1/2 or TLR2/6), as described in further detail below.

TLRs are a conserved family of transmembrane spanning receptors foundprimarily on leukocytes such as dendritic cells (DCs) and macrophages,professional antigen presenting cells. TLRs have specifically evolved torecognize and induce an immune response to pathogen associated molecularpatterns, such as for example bacterial lipoproteins and lipopeptidesand viral double stranded RNA. More than 10 distinct TLRs have beenidentified in mice and humans, although the ligand and signallingpathways are not yet known for some (see Table 1 below). There are 13identified TLRs in humans, numbered 1 through 13.

Type of Adaptor Cellular Agonist Receptor Agonist Molecule LocationExamples TLR1/2 Bacterial MyD88 Surface Pam3Cys lipopeptides TLR3 dsRNATRIF Intracellular Poly I:C TLR4 Lipopoly- MyD88/ Surface LPS, MPLsaccharide TRIF TLR5 Protein MyD88 Surface Flagellin TLR2/6 Bacterialdiacyl MyD88 Surface Zymosan, lipopeptides Pam2Cys TLR7 ssRNA MyD88Intracellular Imiquimod, Loxoribine TLR8 ssRNA, small MyD88Intracellular Resiquimod, synthetic R848 compounds TLR9 UnmethlyatedMyD88 Intracellular CpG DNA

TLRs typically form homodimers, with the exception of TLR2 which forms aheterodimer with TLR1 or TLR6 resulting in differing ligand specificity.TLR2 mediates downstream signalling, so these heterodimers are oftenreferred to collectively as TLR2 (Takeuchi, O. and S. Akira, Cell, 2010,140(6): p. 805-20). Stimulation of the TLRs on DCs results inupregulation of MHC and co-stimulatory molecules, which enhance theantigen presenting function of these cells, as well as the production ofTh1-type cytokines and promotion of cross-presentation (Lahiri et al.,Vaccine, 2008, 26(52): p. 6777-83; Welters et al., Vaccine, 2007, 25(8):p. 1379-89; Matsumoto et al., Adv Drug Deliv Rev, 2008, 60(7): p.805-12; Blander, J. M., Ann Rheum Dis, 2008, 67 Suppl 3: p. iii44-9).Because stimulation through TLRs has a direct effect on boosting theimmune response, TLR agonists have been studied as potential adjuvants(Barchet et al. Curr Opin Immunol, 2008, 20(4): p. 389-95).

TLRs have a conserved cytosolic domain termed the Toll-interleukin 1receptor (TIR) which is associated with an adaptor molecule thatfacilitates downstream signalling pathways leading to cellularactivation. TLRs could be broadly categorized by the adaptor proteinthey are associated with, MyD88 or TRIF. TLR4 alone can signal throughboth pathways. Both signalling pathways converge on the activation ofthe transcription factor NF-KB (Ouyang et al., Biochem Biophys ResCommun, 2007, 354(4): p. 1045-51). Several studies have demonstratedthat although different TLRs share some downstream signalling molecules,each receptor produces a unique profile of pro-inflammatory mediators(Welters et al., Vaccine, 2007, 25(8): p. 1379-89; Seya et al., EvidBased Complement Alternat Med, 2006, 3(1): p. 31-8 and discussion 133-7;Ghosh et al., Cell Immunol, 2006, 243(1): p. 48-57; Re, F. andStrominger. J. L., J Immunol, 2004, 173(12): p. 7548-55; Avril et al., JImmunother, 2009, 32(4): p. 353-62). The full downstream pathway for TLRreceptors are not fully elucidated, but differences in activation couldbe the result of the strength of the ligand, subcellular location of thereceptor, cell type and the presence of interferon regulatory factors(IRF).

Palmitic acid adjuvants have been reported to signal through toll-likereceptor 2 (TLR2). For example, PAM₂Cys is recognized by the heterodimerTLR2 and TLR6. Also as an example, PAM₃Cys, which is recognized by theheterodimer TLR1 and TLR2, triggers an anti-bacterial response typifiedby humoral activity. In contrast double stranded RNA from viruses isrecognized by TLR3 and induces an anti-viral response that is usuallycharacterized by interferon release and T cell activity. Mediatingcellular responses has been associated with TLR2.

Pam₃Cys has been tested in a variety of animal models and in Phase Iclinical trial in humans with no reported side effects (Moyle, P. M. andToth. I., Curr Med Chem, 2008, 15(5): p. 506-16; Wiedemann et al., JPathol, 1991, 164(3): p. 265-71). In a screen of TLR agonists on murineDCs, stimulation with Pam₃Cys in vitro produced high levels of thepro-inflammatory cytokines IL-12p40, IL-6 and TNFα that was attainedwith only small amounts of the adjuvant relative to other TLR agoniststested (Welters et al., Vaccine, 2007, 25(8): p. 1379-89).

As will be appreciated by those skilled in the art, the presentinvention encompasses adjuvants that activate or increase the activityof a TLR, or acts as an agonist to a TLR, particularly a lipid-basedadjuvant. In a particular embodiment, the lipid-based adjuvant activatesor increases the activity of TLR2. Without limitation, such lipid-basedadjuvants may be a palmitic acid adjuvant which activates or increasesthe activity of a TLR, such as a palmitic acid adjuvant comprisingPAM₂Cys or PAM₃Cys.

Other exemplary TLR2 agonists which may be used as a lipid-basedadjuvant in the composition of the invention include, withoutlimitation, cell-wall components such as lipoteichoic acid andlipoprotein from Gram-positive bacteria, and lipoarabinomannan frommycobacteria. A number of these cell-wall components are available fromInvivoGen (San Diego, Calif., USA), such as lipoarabinomannan from M.smegmatis (LAM-MS), lipomannan from M. smegmatis (LM-MS),lipopolysaccharide from P. gingivalis (LPS-PG Ultrapure), andlipoteichoic acid from B. subtilis (LTA-BS) and S. aureus (LTA-SA). Insome embodiments, the lipid-based adjuvant that activates or increasesthe activity of TLR2 may encompass a heat-killed bacteria that comprisesany one or more of the cell-wall components described above. Suchheat-killed bacteria are available, for example, from InvivoGen (SanDiego, Calif., USA).

Synthetic lipoproteins that act as TLR agonists are also encompassed bythe invention, and include without limitation the palmitic acidadjuvants and analogs described above and synthetic diacylatedlipoprotein FSL-1 available from InvivoGen (San Diego, Calif., USA) andEMC Microcollections GmbH (Germany). FSL-1 (Pam2CGDPKHPKSF; SEQ ID NO:2) is a synthetic lipoprotein that represents the N-terminal part of the44-kDa lipoprotein LP44 of Mycoplasma salivarium. FSL-1 comprisesPAM₂Cys and has a similar framework structure as macrophage activatinglipopeptide-2 (MALP-2), a Mycoplasma fermentans derived lipopeptide. Itis postulated that FSL-1 and MALP-2, containing a lipolyated N-terminaldiacylated cysteine residue, are recognized by dimer TLR2 and TLR69TLR2/6). Synthetic MALP-2 is available from Enzo Life Sciences(Farmingdale, N.Y., USA).

In an embodiment, the lipid-based adjuvant of the invention comprisesFSL-1 or MALP-2, or the lipid-based adjuvant is FSL-1 or MALP-2. Whereappropriate, FSL-1 or MALP-2 may used as stereochemically definedcompounds or as a mixture of stereoisomers. The FSL-1 or MALP-2 may belabelled (e.g. biotin, Fluorescein, Rhodamine, etc.). FSL-1 is alsoavailable as a FSL-1 Ala-scan collection (EMC Microcollections)comprising nine different FSL-1-Ala compounds. Each of these FSL-1-Alamolecules is encompassed herein individually or in combination.

Further embodiments of lipid-based adjuvants of the invention mayinclude substructures of TLR2 ligands such as monoacylated lipopeptides.Without limitation, these may include, for example, Pam-Dhc-SKKKK (SEQID NO: 3), Pam-CSKKKK (SEQ ID NO: 1), Pam-Dhc-GDPKHPKSF (SEQ ID NO: 17)or Pam-CGDPKHPKSF (SEQ ID NO: 2) (EMC Microcollections).

Other lipid-based adjuvants that activate or increase the activity ofTLR2 can be identified, for example, by using the InvivoGen (San Diego,Calif., USA) HEK-Blue® TLR2 activation reporter system. This systemallows for evaluation of the ability of potential TLR2 ligands tostimulate TLR2 in either human (hTLR2) or murine (mTLR2) cells.

In some embodiments, the lipid-based adjuvant of the compositions of theinvention is one that activates or increases the activity of only TLR2,heterodimer TLR1 and TLR2 (TLR1/2), and/or heterodimer TLR2 and TLR6(TLR2/6), while other TLRs are not activated. In a further embodiment,the lipid-based adjuvant activates or increases only the activity ofheterodimer TLR1/2 and/or TLR2/6, but does not activate other TLRs.

The composition of the invention may comprise an adjuvant as describedabove in combination with at least one other suitable adjuvant.Exemplary embodiments of the at least one other adjuvant encompasses,but is by no means limited to, organic and inorganic compounds,polymers, proteins, peptides, sugars from synthetic, non-biological orbiological sources (including but not limited to virosomes, virus-likeparticles, viruses and bacteria of their components).

Further examples of compatible adjuvants may include, withoutlimitation, chemokines, Toll like receptor agonists, colony stimulatingfactors, cytokines, 1018 ISS, aluminum salts, Amplivax, AS04, AS15,ABM2, Adjumer, Algammulin, AS01B, AS02 (SBASA). ASO2A, BCG, Calcitriol,Chitosan, Cholera toxin, CP-870,893, CpG, polyIC, CyaA,Dimethyldioctadecylammonium bromide (DDA), Dibutyl phthalate (DBP),dSLIM, Gamma inulin, GM-CSF, GMDP, Glycerol, IC30, IC31, Imiquimod,ImuFact IMP321. IS Patch, ISCOM, ISCOMATRIX, JuvImmune, LipoVac, LPS,lipid core protein, MF59, monophosphoryl lipid A, Montanide® IMS1312,Montanide® based adjuvants, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTelvector system, other palmitoyl based molecules, PLG microparticles,resiquimod, squalene, SLR172, YF-17 DBCG, QS21, QuilA, P1005, Poloxamer,Saponin, synthetic polynucleotides, Zymosan, pertussis toxin.

Accordingly, the composition may comprise one or more pharmaceuticallyacceptable adjuvants, where at least one of the adjuvants of thecomposition is an adjuvant that activates or increases the activity ofTLR2.

In another embodiment, the antigen may be coupled to a lipid moiety,such as for example a palmitic acid moiety, to provide the adjuvantingproperty. The composition may also comprise further pharmaceuticallyacceptable excipients, diluents, etc., as known in the art. See, forexample, Remington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985) and The UnitedStates Pharmacopoeia: The National Formulary (USP 24 NF19) published in1999.

In an embodiment, such additional suitable adjuvants may comprise aCpG-containing oligodeoxynucleotide (CpG ODN), such as5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO: 18). The skilled person mayselect an appropriate CpG on the basis of the target species andefficacy.

The amount of adjuvant used depends on the amount of antigen and on thetype of adjuvant. One skilled in the art can readily determine theamount of adjuvant needed in a particular application by empiricaltesting.

Antigens

The compositions of the invention comprise one or more antigens. As usedherein, the term “antigen” refers to a substance that can bindspecifically to an antibody. Suitable antigens of the composition arethose that are capable of inducing a humoral immune response in asubject.

Antigens useful in the compositions of the invention include, withoutlimitation, polypeptides, carbohydrates, a microorganism or a partthereof, such as a live, attenuated, inactivated or killed bacterium,virus or protozoan, or part thereof. The antigen may be, for example, apathogenic biological agent, a toxin, an allergen, a peptide, a suitablenative, non-native, recombinant or denatured protein or polypeptide, ora fragment thereof, or an epitope that is capable of producing a humoralimmune response in a subject.

As used herein and in the claims, the term “antigen” also includes apolynucleotide that encodes the polypeptide that functions as anantigen. Nucleic acid-based vaccination strategies are known, wherein avaccine composition that contains a polynucleotide is administered to asubject. The antigenic polypeptide encoded by the polynucleotide isexpressed in the subject, such that the antigenic polypeptide isultimately present in the subject, just as if the vaccine compositionitself had contained the polypeptide. For the purposes of the presentinvention, the term “antigen”, where the context dictates, encompassessuch polynucleotides that encode the polypeptide which functions as theantigen.

Polypeptides or fragments thereof that may be useful as antigens in theinvention include, without limitation, those derived from Choleratoxoid, tetanus toxoid, diphtheria toxoid, hepatitis B surface antigen,hemagglutinin (e.g. H5N1 recombinant hemagglutinin protein), anthraxrecombinant protective antigen (List Biologics; Campbell, Calif.),neuraminidase, influenza M protein, PfHRP2, pLDH, aldolase, MSP1, MSP2,AMA1, Der-p-1, Der-f-1, Adipophilin, AFP, AIM-2, ART-4, BAGE, α-fetoprotein, BCL-2, Bcr-Abl, BING-4, CEA, CPSF, CT, cyclin D1Ep-CAM, EphA2,EphA3, ELF-2, FGF-5, G250, Gonadotropin Releasing Hormone, HER-2,intestinal carboxyl esterase (iCE), IL13Rα2, MAGE-1, MAGE-2, MAGE-3,MART-1. MART-2, M-CSF, MDM-2, MMP-2, MUC-1, NY-EOS-1, MUM-1. MUM-2,MUM-3, pertussis toxoid protein, p53, PBF, PRAME, PSA, PSMA, RAGE-1,RNF43, RU1, RU2AS, SART-1, SART-2, SART-3, SAGE-1, SCRN 1, SOX2, SOX10,STEAP1, survivin, Telomerase, TGFβRII, TRAG-3, TRP-1, TRP-2, TERT andWT1.

Viruses, or parts thereof, useful as antigens in the invention include,without limitation, Cowpoxvirus, Vaccinia virus, Pseudocowpox virus,Human herpesvirus 1, Human herpesvirus 2, Cytomegalovirus, Humanadenovirus A-F, Polyomavirus, Human papillomavirus, Parvovirus,Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Humanimmunodeficiency virus, Orthoreovirus, Rotavirus, Ebolavirus,parainfluenza virus, influenza virus (e.g. H5N1 influenza virus,influenza A virus, influenza B virus, influenza C virus). Measles virus,Mumps virus, Rubella virus, Pneumovirus, Human respiratory syncytialvirus, Rabies virus, California encephalitis virus, Japaneseencephalitis virus, Hantaan virus, Lymphocytic choriomeningitis virus,Coronavirus, Enterovirus, Rhinovirus, Poliovirus, Norovirus, Flavivirus,Dengue virus, West Nile virus, Yellow fever virus and varicella.

In an embodiment, a composition of the invention may be used to treatand/or prevent an influenza virus infection in a subject in needthereof. Influenza is a single-stranded RNA virus of the familyOrthomyxoviridae and is often characterized based on two largeglycoproteins on the outside of the viral particle, hemagglutinin (HA)and neuraminidase (NA). Numerous HA subtypes of influenza A have beenidentified (Kawaoka et al., Virology (1990) 179:759-767; Webster et al.,“Antigenic variation among type A influenza viruses,” p. 127-168. In: P.Palese and D. W. Kingsbury (ed.), Genetics of influenza viruses.Springer-Verlag, New York).

Bacteria or parts of thereof useful as antigens in the inventioninclude, without limitation, Anthrax (Bacillus anthracis), Brucella,Bordetella pertussis, Candida, Chlamydia pneumoniae, Chiamydia psittaci,Cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus,Diphtheria, Escherichia coli O157: H7, Enterohemorrhagic Escherichiacoli, Enterotoxigenic Escherichia coli, Haemophilus influenzae,Helicobacter pylori, Legionella, Leptospira, Listeria, Meningococcus,Mycoplasma pneumoniae, Mycobacterium, Pertussis, Pneumonia, Salmonella,Shigella, Staphylococcus, Streptococcus pneumoniae and Yersiniaenterocolitica.

The antigen may alternatively be of protozoan origin, e.g. of the genusPlasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodiumvivax, Plasmodium ovale or Plasmodium knowlesi), which causes malaria.

The antigen may alternatively be a naturally occurring or synthesizedtoxin, such as a drug substance (e.g. cocaine).

The term “polypeptide” encompasses any chain of amino acids, regardlessof length (e.g., at least 6, 8, 10, 12, 14, 16, 18, or 20 amino acids)or post-translational modification (e.g., glycosylation orphosphorylation), and includes, for example, natural proteins, syntheticor recombinant polypeptides and peptides, epitopes, hybrid molecules,variants, homologs, analogs, peptoids, peptidomimetics, etc. A variantor derivative therefore includes deletions, including truncations andfragments; insertions and additions, for example conservativesubstitutions, site-directed mutants and allelic variants; andmodifications, including peptoids having one or more non-amino acylgroups (for example, sugar, lipid, etc.) covalently linked to thepeptide and post-translational modifications. As used herein, the term“conserved amino acid substitutions” or “conservative substitutions”refers to the substitution of one amino acid for another at a givenlocation in the peptide, where the substitution can be made withoutsubstantial loss of the relevant function. In making such changes,substitutions of like amino acid residues can be made on the basis ofrelative similarity of side-chain substituents, for example, their size,charge, hydrophobicity, hydrophilicity, and the like, and suchsubstitutions may be assayed for their effect on the function of thepeptide by routine testing. Specific, non-limiting examples of aconservative substitution include the following examples:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Polypeptides or peptides that have substantial identity to a preferredantigen sequence may be used. Two sequences are considered to havesubstantial identity if, when optimally aligned (with gaps permitted),they share at least approximately 50% sequence identity, or if thesequences share defined functional motifs. In alternative embodiments,optimally aligned sequences may be considered to be substantiallyidentical (i.e., to have substantial identity) if they share at least60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity over aspecified region. The term “identity” refers to sequence similaritybetween two polypeptides molecules. Identity can be determined bycomparing each position in the aligned sequences. A degree of identitybetween amino acid sequences is a function of the number of identical ormatching amino acids at positions shared by the sequences, for example,over a specified region. Optimal alignment of sequences for comparisonsof identity may be conducted using a variety of algorithms, as are knownin the art, including the ClustalW program, available athttp://clustalw.genome.ad.jp, the local homology algorithm of Smith andWaterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithmof Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search forsimilarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci.USA 85:2444, and the computerised implementations of these algorithms(such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, Madison, Wis., U.S.A.).Sequence identity may also be determined using the BLAST algorithm,described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using thepublished default settings). For example, the “BLAST 2 Sequences” tool,available through the National Center for Biotechnology Information(through the internet athttp://www.ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi) may be used,selecting the “blastp” program at the following default settings: expectthreshold 10; word size 3; matrix BLOSUM 62; gap costs existence 11,extension 1. In another embodiment, the person skilled in the art canreadily and properly align any given sequence and deduce sequenceidentity and/or homology by mere visual inspection.

Polypeptides and peptides used to practice the invention can be isolatedfrom natural sources, be synthetic, or be recombinantly generatedpolypeptides. Peptides and proteins can be recombinantly expressed invitro or in vivo. The peptides and polypeptides used to practice theinvention can be made and isolated using any method known in the art.Polypeptide and peptides used to practice the invention can also besynthesized, whole or in part, using chemical methods well known in theart. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223;Hom (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K,Therapeutic Peptides and Proteins, Formulation, Processing and DeliverySystems (1995) Technomic Publishing Co., Lancaster, Pa. For example,peptide synthesis can be performed using various solid-phase techniques(see e.g., Roberge (1995) Science 269:202; Merrifield (1997) MethodsEnzymol. 289:3-13) and automated synthesis may be achieved, e.g., usingthe ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with theinstructions provided by the manufacturer.

In some embodiments, the antigen may be a purified antigen, e.g., fromabout 25% to 50% pure, from about 50% to about 75% pure, from about 75%to about 85% pure, from about 85% to about 90% pure, from about 90% toabout 95% pure, from about 95% to about 98% pure, from about 98% toabout 99% pure, or greater than 99% pure.

As noted above, the term “antigen” also includes a polynucleotide thatencodes the polypeptide that functions as an antigen. As used herein andin the claims, the term “polynucleotide” encompasses a chain ofnucleotides of any length (e.g. 9, 12, 18, 24, 30, 60, 150, 300, 600,1500 or more nucleotides) or number of strands (e.g. single-stranded ordouble-stranded). Polynucleotides may be DNA (e.g. genomic DNA or cDNA)or RNA (e.g. mRNA) or combinations thereof. They may be naturallyoccurring or synthetic (e.g. chemically synthesized). It is contemplatedthat the polynucleotide may contain modifications of one or morenitrogenous bases, pentose sugars or phosphate groups in the nucleotidechain. Such modifications are well-known in the art and may be for thepurpose of e.g. improving stability of the polynucleotide.

The polynucleotide may be delivered in various forms. In someembodiments, a naked polynucleotide may be used, either in linear form,or inserted into a plasmid, such as an expression plasmid. In otherembodiments, a live vector such as a viral or bacterial vector may beused.

One or more regulatory sequences that aid in transcription of DNA intoRNA and/or translation of RNA into a polypeptide may be present. In someinstances, such as in the case of a polynucleotide that is a messengerRNA (mRNA) molecule, regulatory sequences relating to the transcriptionprocess (e.g. a promoter) are not required, and protein expression maybe effected in the absence of a promoter. The skilled artisan caninclude suitable regulatory sequences as the circumstances require.

In some embodiments, the polynucleotide is present in an expressioncassette, in which it is operably linked to regulatory sequences thatwill permit the polynucleotide to be expressed in the subject to whichthe composition of the invention is administered. The choice ofexpression cassette depends on the subject to which the composition isadministered as well as the features desired for the expressedpolypeptide.

Typically, an expression cassette includes a promoter that is functionalin the subject and can be constitutive or inducible; a ribosome bindingsite; a start codon (ATG) if necessary; the polynucleotide encoding thepolypeptide of interest; a stop codon; and optionally a 3′ terminalregion (translation and/or transcription terminator). Additionalsequences such as a region encoding a signal peptide may be included.The polynucleotide encoding the polypeptide of interest may behomologous or heterologous to any of the other regulatory sequences inthe expression cassette. Sequences to be expressed together with thepolypeptide of interest, such as a signal peptide encoding region, aretypically located adjacent to the polynucleotide encoding the protein tobe expressed and placed in proper reading frame. The open reading frameconstituted by the polynucleotide encoding the protein to be expressedsolely or together with any other sequence to be expressed (e.g. thesignal peptide), is placed under the control of the promoter so thattranscription and translation occur in the subject to which thecomposition is administered.

The amount of antigen used in a single treatment with a composition asdescribed herein may vary depending on the type of antigen and the sizeof the subject. One skilled in the art will be able to determine,without undue experimentation, the effective amount of antigen to use ina particular application. The term “effective amount” as used hereinmeans an amount effective, at dosages and for periods of time necessary,to achieve the desired result.

In another embodiment, the antigen may be or comprise a B cell epitopecapable of inducing a humoral immune response. For example, the antigenmay be or comprise a B cell epitope derived from a virus, such as forexample influenza virus or respiratory syncytial virus.

In another embodiment, the B cell epitope may be an epitope derived fromthe hemagglutinin glycoprotein of the H5N1 influenza virus.

In another embodiment, the antigen may be or comprise a B cell epitopederived from a bacterium, such as for example Bordetella pertussis orBacillus anthracis.

In another embodiment, the B cell epitope may be an epitope of thepertussis toxoid protein produced by Bordetella pertussis.

In another embodiment, the B cell epitope may be an epitope of theanthrax recombinant protective antigen.

In another embodiment, the antigen may be or comprise a B cell epitopeassociated with an infectious disease.

In another embodiment, the antigen may be or comprise a B cell epitopederived from a protozoan, such as from the genus Plasmodium.

In another embodiment, the antigen may be a cancer or tumor-associatedprotein, such as for example, a membrane surface-bound cancer antigenwhich is capable of being recognized by an antibody.

Cancers that may be treated and/or prevented by the use oradministration of a composition of the invention include, withoutlimitation, carcinoma, adenocarcinoma, lymphoma, leukemia, sarcoma,blastoma, myeloma, and germ cell tumors. In one embodiment, the cancermay be caused by a pathogen, such as a virus. Viruses linked to thedevelopment of cancer are known to the skilled person and include, butare not limited to, human papillomaviruses (HPV), John Cunningham virus(JCV), Human herpes virus 8, Epstein Barr Virus (EBV), Merkel cellpolyomavirus, Hepatitis C Virus and Human T cell leukaemia virus-1. Acomposition of the invention may be used for either the treatment orprophylaxis of cancer, for example, in the reduction of the severity ofcancer or the prevention of cancer recurrences. Cancers that may benefitfrom the compositions of the invention include any malignant cell thatexpresses one or more tumor specific antigens.

In another embodiment, the antigen may be a toxin or an allergen that iscapable of being neutralized by an antibody. In an embodiment, the toxinis a drug substance such as, for example, cocaine.

In another embodiment, the antigen may be an antigen associated with adisease where it is desirable to sequester the antigen in circulation,such as for example an amyloid protein (e.g. Alzheimer's disease). Thus,a composition of the invention may be suitable for use in the treatmentand/or prevention of a neurodegenerative disease in a subject in needthereof, wherein the neurodegenerative disease is associated with theexpression of an antigen. The subject may have a neurodegenerativedisease or may be at risk of developing a neurodegenerative disease.Neurodegenerative diseases that may be treated and/or prevented by theuse or administration of a composition of the invention include, withoutlimitation, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, and amyotrophic lateral sclerosis (ALS). For example,

Alzheimer's disease is characterized by the association of B-amyloidplaques and/or tau proteins in the brains of patients with Alzheimer'sdisease (see, for example, Goedert and Spillantini, Science, 314:777-781, 2006). Herpes simplex virus type 1 has also been proposed toplay a causative role in people carrying the susceptible versions of theapoE gene (Itzhaki and Wozniak, J Alzheimers Dis 13: 393-405, 2008).

In a further embodiment, the composition may comprise a mixture of Bcell epitopes as antigens for inducing a humoral immune response. The Bcell epitopes may be linked to form a single polypeptide.

In another embodiment, the antigen may be any peptide or polypeptidethat is capable of inducing a specific humoral immune response to aspecific conformation on targeted tumor cells.

T Helper Epitopes

T helper epitopes are a sequence of amino acids (natural or non-naturalamino acids) that have T helper activity. T helper epitopes arerecognised by T helper lymphocytes, which play an important role inestablishing and maximising the capabilities of the immune system, andare involved in activating and directing other immune cells, such as forexample B cell antibody class switching.

A T helper epitope can consist of a continuous or discontinuous epitope.Hence not every amino acid of a T helper is necessarily part of theepitope. Accordingly, T helper epitopes, including analogs and segmentsof T helper epitopes, are capable of enhancing or stimulating an immuneresponse. Immunodominant T helper epitopes are broadly reactive inanimal and human populations with widely divergent MHC types (Celis etal. (1988) J. Immunol. 140:1808-1815: Demotz et al. (1989) J. Immunol.142:394-402; Chong et al. (1992) Infect. Immun. 60:4640-4647). The Thelper domain of the subject peptides has from about 10 to about 50amino acids and preferably from about 10 to about 30 amino acids. Whenmultiple T helper epitopes are present, then each T-helper epitope actsindependently.

In one embodiment, the composition described herein may also comprise atleast one T helper epitope. In some instances, the T-helper epitope mayform part of the antigen. In particular, if the antigen is of sufficientsize, it may contain an epitope that functions as a T-helper epitope. Inother embodiments, the T-helper epitope is a separate molecule from theantigen.

In another embodiment, T helper epitope analogs may includesubstitutions, deletions and insertions of from one to about 10 aminoacid residues in the T helper epitope. T helper segments are contiguousportions of a T helper epitope that are sufficient to enhance orstimulate an immune response. An example of T-helper segments is aseries of overlapping peptides that are derived from a single longerpeptide.

Sources of T helper epitopes for use in the present invention include,for example, hepatitis B surface antigen helper T cell epitopes,pertussis toxin helper T cell epitopes, measles virus F protein helper Tcell epitope, Chlamydia trachomitis major outer membrane protein helperT cell epitope, diphtheria toxin helper T cell epitopes, Plasmodiumfalciparum circumsporozoite helper T cell epitopes, Schistosoma mansonitriose phosphate isomerase helper T cell epitopes, Escherichia coli TraThelper T cell epitopes and immune-enhancing analogs and segments of anyof these T helper epitopes.

In one embodiment, the T helper epitope is a universal T helper epitope.A universal T helper epitope as used herein refers to a peptide or otherimmunogenic molecule, or a fragment thereof, that binds to amultiplicity of MHC class II molecules in a manner that activates T-cellfunction in a class II (CD4⁺ T cells)-restricted manner.

In another embodiment, the T helper epitope may be a universal T helperepitope such as PADRE (pan-DR epitope) comprising the peptide sequenceAKXVAAWTLKAAA (SEQ ID NO: 19), wherein X may be cyclohexylalanyl. PADREspecifically has a CD4⁺ T-helper epitope, that is, it stimulatesinduction of a PADRE-specific CD4 T helper response.

Tetanus toxoid has T helper epitopes that work in the similar manner asPADRE. Tetanus and diphtheria toxins have universal epitopes for humanCD4⁺ cells. (Diethelm-Okita, B. M. et al., Universal epitopes for humanCD⁴⁺ cells on tetanus and diphtheria toxins. J. Infect. Diseases,181:1001-1009, 2000). In another embodiment, the T helper epitope may bea tetanus toxoid peptide such as F21E comprising the peptide sequenceFNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 20) (amino acids 947-967).

In another embodiment, the T helper epitope is fused to at least oneantigen (i.e., a peptide), or a mixture of antigens, to make a fusionpeptide.

Carriers

The carrier of the composition comprises a continuous phase of ahydrophobic substance, preferably a liquid hydrophobic substance. Thecontinuous phase may be an essentially pure hydrophobic substance or amixture of hydrophobic substances. In addition, the carrier may be anemulsion of water in a hydrophobic substance or an emulsion of water ina mixture of hydrophobic substances, provided the hydrophobic substanceconstitutes the continuous phase. Further, in another embodiment, thecarrier may function as an adjuvant.

Hydrophobic substances that are useful in the compositions as describedherein are those that are pharmaceutically and/or immunologicallyacceptable. The carrier is preferably a liquid but certain hydrophobicsubstances that are not liquids at atmospheric temperature may beliquefied, for example by warming, and are also useful in thisinvention. In one embodiment, the hydrophobic carrier may be a PhosphateBuffered Saline/Freund's Incomplete Adjuvant (PBS/FIA) emulsion.

Oil or water-in-oil emulsions are particularly suitable carriers for usein the present invention. Oils should be pharmaceutically and/orimmunologically acceptable. Suitable oils include, for example, mineraloils (especially light or low viscosity mineral oil such as Drakeo®6VR), vegetable oils (e.g., soybean oil), nut oils (e.g., peanut oil),or mixtures thereof. In an embodiment, the oil is a mannide oleate inmineral oil solution, commercially available as Montanide® ISA 51.Animal fats and artificial hydrophobic polymeric materials, particularlythose that are liquid at atmospheric temperature or that can beliquefied relatively easily, may also be used.

In embodiments herein where the composition is described as being awater-free liposome suspension (“water-free”), it is possible that thehydrophobic carrier of these “water-free”compositions may still containsmall quantities of water, provided that the water is present in thenon-continuous phase of the carrier. For example, individual componentsof the composition may have bound water that may not be completelyremoved by processes such as lyophilization or evaporation and certainhydrophobic carriers may contain small amounts of water dissolvedtherein. Generally, compositions of the invention that are described as“water-free” contain, for example, less than about 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% water on a weight/weightbasis of the total weight of the carrier component of the composition.

Liposomes

Liposomes are completely closed lipid bilayer membranes containing anentrapped aqueous volume. Liposomes may be unilamellar vesicles(possessing a single bilayer membrane) or multilamellar vesiclescharacterized by multimembrane bilayers, each bilayer may or may not beseparated from the next by an aqueous layer. A general discussion ofliposomes can be found in Gregoriadis G. Immunol. Today, 11:89-97, 1990;and Frezard, F., Braz. J. Med. Bio. Res., 32:181-189, 1999. As usedherein and in the claims, the term “liposomes” is intended to encompassall such vesicular structures as described above, including, withoutlimitation, those described in the art as “niosomes”, “transfersomes”and “virosomes”.

Although any liposomes may be used in this invention, includingliposomes made from archaebacterial lipids, particularly usefulliposomes use phospholipids and unesterified cholesterol in the liposomeformulation. The cholesterol is used to stabilize the liposomes and anyother compound that stabilizes liposomes may replace the cholesterol.Other liposome stabilizing compounds are known to those skilled in theart. For example, saturated phospholipids produce liposomes with highertransition temperatures indicating increased stability.

Phospholipids that are preferably used in the preparation of liposomesare those with at least one head group selected from the groupconsisting of phosphoglycerol, phosphoethanolamine, phosphoserine,phosphocholine and phosphoinositol. More preferred are liposomes thatcomprise lipids which are 94-100% phosphatidylcholine. Such lipids areavailable commercially in the lecithin Phospholipon® 90 G. Whenunesterified cholesterol is also used in liposome formulation, thecholesterol is used in an amount equivalent to about 10% of the weightof phospholipid. If a compound other than cholesterol is used tostabilize the liposomes, one skilled in the art can readily determinethe amount needed in the composition.

Liposome compositions may be obtained, for example, by using naturallipids, synthetic lipids, sphingolipids, ether lipids, sterols,cardiolipin, cationic lipids and lipids modified with poly (ethyleneglycol) and other polymers. Synthetic lipids may include the followingfatty acid constituents; lauroyl, myristoyl, palmitoyl, stearoyl,arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these fattyacids.

Compositions

Further embodiments of the present invention include methods of making acomposition of the invention comprising liposomes; an antigen capable ofinducing a humoral immune response; a carrier comprising a continuousphase of a hydrophobic substance; and an adjuvant that activates orincreases the activity of TLR2.

Methods for making liposomes are well known in the art. See e.g.Gregoriadis (1990) and Frezard (1999) both cited previously. Anysuitable method for making liposomes may be used in the practice of theinvention, or liposomes may be obtained from a commercial source.Liposomes are typically prepared by hydrating the liposome componentsthat will form the lipid bilayer (e.g. phospholipids and cholesterol)with an aqueous solution, which may be pure water or a solution of oneor more components dissolved in water, e.g. phosphate-buffered saline(PBS), phosphate-free saline, or any other physiologically compatibleaqueous solution.

In an embodiment, a liposome component or mixture of liposomecomponents, such as a phospholipid (e.g. Phospholipon® 90G) andcholesterol, may be solubilized in an organic solvent, such as a mixtureof chloroform and methanol, followed by filtering (e.g. a PTFE 0.2 μmfilter) and drying, e.g. by rotary evaporation, to remove the solvents.

Hydration of the resulting lipid mixture may be effected by e.g.injecting the lipid mixture into an aqueous solution or sonicating thelipid mixture and an aqueous solution. During formation of liposomes,the liposome components form single bilayers (unilamellar) or multiplebilayers (multilamellar) surrounding a volume of the aqueous solutionwith which the liposome components are hydrated.

In some embodiments, the liposomes are then dehydrated, such as byfreeze-drying or lyophilization.

The liposomes are combined with the carrier comprising a continuoushydrophobic phase. This can be done in a variety of ways.

If the carrier is composed solely of a hydrophobic substance or amixture of hydrophobic substances (e.g. use of a 100% mineral oilcarrier), the liposomes may simply be mixed with the hydrophobicsubstance, or if there are multiple hydrophobic substances, mixed withany one or a combination of them.

If instead the carrier comprising a continuous phase of a hydrophobicsubstance contains a discontinuous aqueous phase, the carrier willtypically take the form of an emulsion of the aqueous phase in thehydrophobic phase, such as a water-in-oil emulsion. Such compositionsmay contain an emulsifier to stabilize the emulsion and to promote aneven distribution of the liposomes. In this regard, emulsifiers may beuseful even if a water-free carrier is used, for the purpose ofpromoting an even distribution of the liposomes in the carrier. Typicalemulsifiers include mannide oleate (Arlacel™ A), lecithin (e.g. S100lecithin), a phospholipid, Tween™80, and Spans™ 20, 80, 83 and 85.Typically, the volume ratio (v/v) of hydrophobic substance to emulsifieris in the range of about 5:1 to about 15:1 with a ratio of about 10:1being preferred.

The liposomes may be added to the finished emulsion, or they may bepresent in either the aqueous phase or the hydrophobic phase prior toemulsification.

The antigen may be introduced at various different stages of theformulation process. More than one type of antigen may be incorporatedinto the composition (e.g. an inactivated virus, attenuated live virus,protein or polypeptide).

In some embodiments, the antigen is present in the aqueous solution usedto hydrate the components that are used to form the lipid bilayers ofthe liposomes (e.g. phospholipid(s) and cholesterol). In this case, theantigen will be encapsulated in the liposome, present in its aqueousinterior. If the resulting liposomes are not washed or dried, such thatthere is residual aqueous solution present that is ultimately mixed withthe carrier comprising a continuous phase of a hydrophobic substance, itis possible that additional antigen may be present outside the liposomesin the final product. In a related technique, the antigen may be mixedwith the components used to form the lipid bilayers of the liposomes,prior to hydration with the aqueous solution. The antigen may also beadded to pre-formed liposomes, in which case the antigen may be activelyloaded into the liposomes, or bound to the surface of the liposomes orthe antigen may remain external to the liposomes. In such embodiments,prior to the addition of antigen, the pre-formed liposomes may be emptyliposomes (e.g. not containing encapsulated antigen or lipid-basedadjuvant) or the pre-formed liposomes may contain lipid-based adjuvantincorporated into or associated with the liposomes. These steps maypreferably occur prior to mixing with the carrier comprising acontinuous phase of a hydrophobic substance.

In an alternative approach, the antigen may instead be mixed with thecarrier comprising a continuous phase of a hydrophobic substance,before, during, or after the carrier is combined with the liposomes. Ifthe carrier is an emulsion, the antigen may be mixed with either or bothof the aqueous phase or hydrophobic phase prior to emulsification.Alternatively, the antigen may be mixed with the carrier afteremulsification.

The technique of combining the antigen with the carrier may be usedtogether with encapsulation of the antigen in the liposomes as describedabove, such that antigen is present both within the liposomes and in thecarrier comprising a continuous phase of a hydrophobic substance.

The above-described procedures for introducing the antigen into thecomposition apply also to the adjuvant of the compositions of thepresent invention. That is, the adjuvant may be introduced into e.g. anyone or more of: (1) the aqueous solution used to hydrate the componentsthat are used to form the lipid bilayers of the liposomes; (2) theaqueous solution after formation of the lipid bilayers of the liposomes;(3) the components used to form the lipid bilayers of the liposomes; or(4) the carrier comprising a continuous phase of a hydrophobicsubstance, before, during, or after the carrier is combined with theliposomes. If the carrier is an emulsion, the adjuvant may be mixed witheither or both of the aqueous phase or hydrophobic phase before, duringor after emulsification.

The technique of combining the adjuvant with the carrier may be usedtogether with encapsulation of the adjuvant in the liposomes, or withaddition of the adjuvant to the liposomes, such that adjuvant is presentinside and/or outside the liposomes and in the carrier comprising acontinuous phase of a hydrophobic substance.

The adjuvant can be incorporated in the composition together with theantigen at the same processing step, or separately, at a differentprocessing step. For instance, the antigen and the adjuvant may both bepresent in the aqueous solution used to hydrate the lipidbilayer-forming liposome components, such that both the antigen andadjuvant become encapsulated in the liposomes. Alternatively, theantigen may be encapsulated in the liposomes, and the adjuvant mixedwith the carrier comprising a continuous phase of a hydrophobicsubstance. In a further embodiment, the adjuvant may be incorporatedinto the composition after the antigen encapsulation step by passing theliposome-antigen preparation through a manual mini-extruder and thenmixing the obtained liposome-antigen preparation with the lipid-basedadjuvant in, for example, phosphate buffer. The adjuvant may also beincorporated into the composition, either alone or together withantigen, after the liposomes have been formed, such that the adjuvantmay be associated or remain external to the liposomes. The adjuvant mayalso be incorporated into or associated with liposomes prior to additionof antigen, with the antigen remaining outside the pre-formed liposomesor loaded into/associated with the liposomes by further processing. Insuch embodiments, the resulting liposome-antigen-adjuvant preparationmay by lyophilized and then reconstituted in the carrier comprising acontinuous phase of a hydrophobic substance. It will be appreciated thatmany such combinations are possible.

If the composition contains one or more further adjuvants, suchadditional adjuvants can be incorporated in the composition in similarfashion as described above for the adjuvant or by combining several ofsuch methods as may be suitable for the additional adjuvant(s).

Stabilizers such as sugars, anti-oxidants, or preservatives thatmaintain the biological activity or improve chemical stability toprolong the shelf life of antigen, adjuvant, the liposomes or thecontinuous hydrophobic carrier, may be added to such compositions.

In some embodiments, an antigen/adjuvant mixture may be used, in whichcase the antigen and adjuvant are incorporated into the composition atthe same time. An “antigen/adjuvant mixture” refers to an embodiment inwhich the antigen and adjuvant are in the same diluent at least prior toincorporation into the composition. The antigen and adjuvant in anantigen/adjuvant mixture may, but need not necessarily be chemicallylinked, such as by covalent bonding.

In some embodiments, the carrier comprising a continuous phase of ahydrophobic substance may itself have adjuvanting-activity. IncompleteFreund's adjuvant, is an example of a hydrophobic carrier withadjuvanting effect. As used herein and in the claims, when the term“adjuvant” is used, this is intended to indicate the presence of anadjuvant in addition to any adjuvanting activity provided by the carriercomprising a continuous phase of a hydrophobic substance.

In an embodiment, to formulate a composition of the invention, ahomogenous mixture of S100 lecithin and cholesterol (e.g. 10:1 w:w) arehydrated in the presence of an antigen, optionally in phosphate buffer,to form liposomes with encapsulated antigen. The liposome preparationmay then be extruded, optionally through a manual mini-extruder, andmixed with the adjuvant, optionally in phosphate buffer, to incorporatethe adjuvant. This suspension may then be lyophilized and reconstitutedin a carrier comprising a continuous phase of a hydrophobic substance toform a water-free liposome suspension.

In some embodiments, the composition may be formulated by hydrating ahomogenous mixture of S100 lecithin and cholesterol (e.g. 10:1 w:w) inthe presence of an antigen and a suitable adjuvant (e.g. Pam-3-Cys),optionally in phosphate buffer, to form liposomes with encapsulatedantigen and adjuvant. The liposome/antigen/adjuvant preparation may thenbe diluted to sufficient quantity, optionally using water, andlyophilized. The lyophilized liposomes may then be reconstituted in acarrier comprising a continuous phase of a hydrophobic substance (e.g.mineral oil or Montanide® ISA 51) to form a water-free liposomesuspension.

In some embodiments, the composition may be formulated by hydrating ahomogenous mixture of dioleoyl-phosphatidylcholine (DOPC) andcholesterol (e.g. 10:1 w:w) in the presence of an antigen and a suitableadjuvant (e.g. Pam-3-Cys-Ser-(Lys)4; SEQ ID NO: 1), optionally inphosphate buffer, to form liposomes encapsulated with antigen andadjuvant. The liposome/antigen/adjuvant preparation may then belyophilized and the resultant product reconstituted in a carriercomprising a continuous phase of a hydrophobic substance (e.g. mineraloil or Montanide® ISA 51) to form a water-free liposome suspension.

Alternatively, the antigen or antigen/adjuvant complex may be associatedwith, in contact with or separate from liposomes and not encapsulated inliposomes. The efficiency of liposome encapsulation of some hydrophilicantigens or hydrophilic antigen/adjuvant complexes may be poor so thatupon being placed in a hydrophobic environment or freeze-drying most ofthe antigen becomes associated with the external surface of theliposomes. This represents another embodiment of the invention.

In a further embodiment, an antigen (peptide or polypeptide) having a Bcell epitope and PADRE (fused to the antigen or separate) may beencapsulated together in liposomes. In another embodiment, more than oneantigen may be placed together in the same liposomes. In a furtherembodiment, other substances may be used instead of PADRE that have aT-helper epitope, for example, tetanus toxoid peptide(s). In anotherembodiment, a adjuvant, preferably a palmitic acid based adjuvant whichcomprises PAM₂Cys or PAM₃Cys, may be encapsulated in the liposomes aswell. The liposomes are preferably suspended in PBS. This suspension isthen emulsified in a hydrophobic carrier, such as for example, ISA51 ormineral oil. The result is that liposomes containing the antigen(s) andadjuvant(s) are suspended in PBS which in turn is emulsified in ahydrophobic carrier, for example, ISA51 or mineral oil.

In one embodiment, antibody titers obtained from mice injectedintramuscularly with a single dose of a composition of the inventioncomprising liposomes/H5N1 recombinant hemagglutinin protein(antigen)/Pam-3-Cys-Ser-(Lys)4 (SEQ ID NO: 1) (adjuvant)/hydrophobiccarrier (Vaccine A) were significantly enhanced at three, four and eightweeks post-immunization compared to mice treated (and boosted) with anaqueous aluminum based control vaccine (FIG. 1). For example, Vaccine Aof the invention was capable of generating endpoint antibody titers atthree and four weeks post-vaccination of up to 1/2,048,000 and up to1/8,192,000 at eight weeks post-vaccination, whereas endpoint antibodytiters for control vaccine were only up to 1/512,000, 1/256,000 and1/4,096,000 at three, four and eight weeks post-vaccination,respectively. These results indicate that compositions of the inventionare capable of generating, upon single dose, an enhanced in vivo humoralimmune response compared to single or boosted aqueous alum based controlvaccine.

In one embodiment, immunization of mice by single treatment with acomposition of the invention comprising liposomes/pertussis toxoidprotein (antigen)/Pam-3-Cys-Ser-(Lys)4 (SEQ ID NO: 1)(adjuvant)/hydrophobic carrier (Vaccine B) was able to reduce bacteriallung counts from as high as 6.2×10^4 cfu per lung at day 8post-challenge with Bordetella pertussis to 0 cfu per lung at day 15post-challenge (FIG. 2). These results indicate that a single dose of acomposition of the invention effectively protects mice from bacterialchallenge and allows them to completely clear the infection from thelungs.

In one embodiment, antibody titers obtained from rabbits injectedintramuscularly with a single dose of a composition of the inventioncomprising liposomes/anthrax recombinant protective antigen(antigen)/Pam-3-Cys (adjuvant)/hydrophobic carrier (Vaccine C) weresignificantly enhanced compared to rabbits treated with an aqueousaluminum based control vaccine at the early (pre-boost) time points(FIG. 3). For example, Vaccine C of the invention was capable ofgenerating endpoint antibody titers at three and four weekspost-vaccination of up to 1/2,048,000 and up to 1/8,192,000 at eightweeks post-vaccination, whereas endpoint antibody titers for controlvaccine were only up to 1/64,000, 1/256,000 and 1/2,048,000 at three,four and eight weeks post-vaccination, respectively. These resultsindicate that compositions of the invention are capable of generating asurprisingly strong in vivo humoral immune response as early as threeweeks following a single vaccination.

In one embodiment, antibody titres obtained from mice injectedintramuscularly with a single dose of Vaccine A of the invention(Group 1) were significantly enhanced compared to single doseadministration of control compositions without liposomes (Group 2),without hydrophobic carrier (Group 3) or without lipid-based adjuvant(Group 4) (FIG. 4). For example, Vaccine C of the invention was capableof generating endpoint antibody titers at eight weeks post-vaccinationof up to 1/2,048,000, while Groups 2, 3 and 4 were only able to generateendpoint antibody titers at the same time point of 1/64,000, 1/128,000and 1/128,000, respectively. These results show the that compositions ofthe invention comprising each of: an antigen, liposomes, a lipid-basedadjuvant and a carrier comprising a continuous phase of a hydrophobicsubstance, are capable of raising robust and long lasting in vivohumoral immune responses.

The compositions as described herein may be formulated in a form that issuitable for oral, nasal, rectal or parenteral administration.Parenteral administration includes intravenous, intraperitoneal,intradermal, subcutaneous, intramuscular, transepithelial,intrapulmonary, intrathecal, and topical modes of administration. Thepreferred routes are intramuscular, subcutaneous and intradermal toachieve a depot effect.

The skilled artisan can determine suitable treatment regimes, routes ofadministration, dosages, etc., for any particular application in orderto achieve the desired result. Factors that may be taken into accountinclude, e.g.: the nature of the antigen; the disease state to beprevented or treated; the age, physical condition, body weight, sex anddiet of the subject; and other clinical factors. See, for example,“Vaccine Handbook”, edited by the Researcher's Associates (Gaku-yuu-kai)of The National Institute of Health (1994); “Manual of ProphylacticInoculation, 8th edition”, edited by Mikio Kimura, Munehiro Hirayama,and Harumi Sakai, Kindai Shuppan (2000); “Minimum Requirements forBiological Products”, edited by the Association of BiologicalsManufacturers of Japan (1993).

The optimal amount of adjuvant and antigen to elicit an optimal immuneresponse may depend on a number of factors including, withoutlimitation, the composition, the disease, the subject, and may bereadily ascertained by the skilled person using standard studiesincluding, for example, observations of antibody titers and otherimmunogenic responses in the host.

The compositions as described herein may be effective when administeredin a single application.

In another embodiment, the compositions as described herein may be usedin combination, before or after, with other therapies.

Kits and Reagents

The present invention is optionally provided to a user as a kit. Forexample, a kit of the invention contains one or more of the compositionsof the invention. The kit can further comprise one or more additionalreagents, packaging material, containers for holding the components ofthe kit, and an instruction set or user manual detailing preferredmethods of using the kit components.

EMBODIMENTS OF THE INVENTION

Particular embodiments of the invention include, without limitation, thefollowing:

1. A composition comprising, consisting of, or consisting essentiallyof: liposomes; an antigen capable of inducing a humoral immune response;a carrier comprising a continuous phase of a hydrophobic substance; andan adjuvant that activates or increases the activity of TLR2, preferablya lipid-based adjuvant.

2. The composition of paragraph 1, wherein the adjuvant activates orincreases the activity of toll-like receptor 2 (TLR2), or a TLR2 dimersuch as TLR1/2 or TLR2/6.

3. The composition of paragraph 1, wherein the adjuvant only activatesor increases the activity of a toll-like receptor (TLR) selected fromTLR2, heterodimeric TLR1/2 and heterodimeric TLR2/6, but does notactivate or increase the activity of other TLRs.

4. The composition of any one of paragraphs 1 to 3, wherein the adjuvantis a compound comprising, consisting of, or consisting essentially of atleast one natural, synthetic or semi-synthetic lipid moiety, lipidcomponent, or analog or derivative thereof, including for example alipoamino acid, a lipoglycan, a lipopolysaccharide, a lipoteichoic acidor a cell-wall component of a Gram-positive or Gram-negative bacteria,Rhodopseudomonas viridis or mycoplasma.

5. The composition of any one of paragraphs 1 to 4, wherein the adjuvantcomprises, consists of, or consists essentially of PAM₂Cys-Ser-(Lys)4(SEQ ID NO: 1), PAM₃Cys-Ser-(Lys)4 (SEQ ID NO: 1), PAM₃Cys-SKKKK (SEQ IDNO: 1)(β-irradiated), R-PAM₃Cys-SKKKK (SEQ ID NO: 1), S-PAM₃Cys-SKKKK(SEQ ID NO: 1), PAM₃Cys-SKKKK(Biotin-Aca-Aca) (SEQ ID NO: 1),PAM₃Cys-SKKKK(Fluorescein-Aca-Aca) (SEQ ID NO: 1), PAM₃Cys-SKKKK(Rhodamine-Aca-Aca) (SEQ ID NO: 1), PAM₃Cys-SKKKK-FLAG-tag (SEQ ID NO:1), PHC-SKKKK (SEQ ID NO: 3), PHC-SKKKK(Biotin-Aca-Aca) (SEQ ID NO: 3),PAM₃Cys-SSNAKIDQLSSDVQT (SEQ ID NO: 4), PAM₃Cys-SSNKSTTGSGETTTA (SEQ IDNO: 5), PAM₃Cys-SSTKPVSQDTSPKPA (SEQ ID NO: 6), PAM₃Cys-SSGSKPSGGPLPDAK(SEQ ID NO: 7), PAM₃Cys-SSGNKSAPSSSASSS (SEQ ID NO: 8),PAM₃Cys-GSHQMKSEGHANMQL (SEQ ID NO: 9), PAM₃Cys-SSSNNDAAGNGAAQT (SEQ IDNO: 10), PAM₃Cys-KQNVSSLDEKNSVSV (SEQ ID NO: 11),PAM₃Cys-NNSGKDGNTSANSAD (SEQ ID NO: 12), PAM₃Cys-NNGGPELKSDEVAKS (SEQ IDNO: 13), PAM₃Cys-SQEPAAPAAEATPAG (SEQ ID NO: 14),PAM₃Cys-SSSKSSDSSAPKAYG (SEQ ID NO: 15), PAM₃Cys-AQEKEAKSELDYDQT (SEQ IDNO: 16), PAM₂Cys-SKKKK (mixture of RR and RS stereoisomers), (SEQ IDNO: 1) R-PAM₂Cys-SKKKK (RR stereoisomer) (SEQ ID NO: 1), S-PAM₂Cys-SKKKK(RS stereoisomer) (SEQ ID NO: 1), PAMCys(PAM)-SKKKK,PAM₂Cys-SKKKK(Biotin-Aca-Aca)-NH₂ (SEQ ID NO: 1), PAM₂Cys-SKKKK(Fluorescein-Aca-Aca)-NH₂ (SEQ ID NO: 1),PAM₂Cys-SKKKK(Rhodamine-Aca-Aca)-NH₂ (SEQ ID NO: 1),PAM₂Cys-SKKKK-FLAG-tag (SEQ ID NO: 1), PAM-Dhc-SKKKK (SEQ ID NO: 3),PAM-CSKKKK (SEQ ID NO: 1), PAM-Dhc-GDPKHPKSF (SEQ ID NO: 17),PAM-CGDPKHPKSF (SEQ ID NO: 2), FSL-1 (Pam2CGDPKHPKSF; SEQ ID NO: 2),FSL-1-Ala, macrophage activating lipopeptide-2 (MALP-2),lipoarabinomannan from M. smegmatis (LAM-MS), lipomannan from M.smegmatis (LM-MS), lipopolysaccharide from P. gingivalis (LPS-PGUltrapure), lipoteichoic acid from B. subtilis (LTA-BS) or S. aureus(LTA-SA), or derivatives or analogs thereof, or is a heat-killedbacteria that comprises the cell-wall component of a Gram-positive orGram-negative bacteria.

6. The composition of any one of paragraphs 1 to 5, wherein theadjuvantis a palmitic acid adjuvant.

7. The composition of any one of paragraphs 1 to 6, wherein the adjuvantcomprises, consists of, or consists essentially ofdipalmitoyl-S-glyceryl-cysteine (PAM₂Cys) ortripalmitoyl-S-glyceryl-cysteine (PAM₃Cys).

8. The composition of any one of paragraphs 1 to 6, wherein the adjuvantis PAM₂Cys-Ser-(Lys)4 (SEQ ID NO: 1), PAM₃Cys-Ser-(Lys)4 (SEQ ID NO: 1),FSL-1 or MALP-2.

9. The composition of paragraph 8, wherein the adjuvant isPAM₂Cys-Ser-(Lys)4 (SEQ ID NO: 1).

10. The composition of paragraph 8, wherein the adjuvant isPAM₃Cys-Ser-(Lys)4 (SEQ ID NO: 1).

11. The composition of any one of paragraphs 1 to 10 which furthercomprises at least one other suitable adjuvant in addition to theadjuvant that activates or increases the activity of TLR2.

12. The composition of any one of paragraphs 1 to 11, wherein theantigen is a polypeptide or a carbohydrate.

13. The composition of any one of paragraphs 1 to 12, wherein theantigen comprises, consists of, or consists essentially of a B cellepitope, or a plurality of B cell epitopes.

14. The composition of paragraph 13, wherein the B cell epitope isderived from a virus or bacteria.

15. The composition of paragraph 14, wherein the B cell epitope isderived from influenza virus, Bordetella pertussis or Bacillusanthracis.

16. The composition of paragraph 15, wherein the B cell epitope is anepitope of a hemagglutinin protein of H5N1 influenza virus, an epitopeof pertussis toxoid protein, or an epitope of an anthrax recombinantprotective antigen.

17. The composition of any one of paragraphs 1 to 13, wherein theantigen is:

-   -   an antigen associated with an infectious disease; a membrane        surface-bound cancer antigen; a toxin; an allergen such as        pollen; or an antigen associated with a disease where it is        desirable to sequester the antigen in circulation, such as for        example an amyloid protein.

18. The composition of paragraph 17, wherein the infectious disease isinfluenza, a respiratory tract infection caused by human respiratorysyncytial virus, pertussis, anthrax or malaria.

19. The composition of paragraph 17, wherein the toxin is a drugsubstance, such as cocaine.

20. The composition of any one of paragraphs 1 to 13, wherein theantigen is a hapten-carrier adduct.

21. The composition of any one of paragraphs 1 to 20, wherein theliposome comprises, consists of, or consists essentially of aphospholipid or unesterified cholesterol.

22. The composition of any one of paragraphs 1 to 21, wherein theantigen is encapsulated in the liposomes or both the antigen and theadjuvant are encapsulated in the liposomes.

23. The composition of any one of paragraphs 1 to 22, which is awater-free liposome suspension.

24. The composition of any one of paragraphs 1 to 23, wherein thecomposition is capable of inducing a humoral immune response with asingle dose.

25. The composition of paragraph 24, wherein the humoral immune responseis characterized by antigen-specific antibody production.

26. The composition of paragraph 25 which is capable of generating theantigen-specific antibody at an antibody titer of up to about1/2,048,000 by about three weeks post-vaccination of a subject.

27. The composition of paragraph 25 which is capable of generating theantigen-specific antibody at an antibody titer of up to about1/8,192,000 by about eight weeks post-vaccination of a subject.

28. The composition of any one of paragraphs 24 to 27, wherein thehumoral immune response is associated with the activation or generationof T-helper 2 (Th2) cells or T-helper 17 (Th17) cells.

29. The composition of any one of paragraphs 1 to 28 for the treatmentor prevention of a disease or disorder ameliorated by a humoral immuneresponse.

30. The composition of any one of paragraphs 1 to 28 for the treatmentor prevention of: an infectious disease; a cancer involving a membranesurface-bound cancer antigen; or a disease or disorder where it isdesirable to sequester antigen in circulation, such as Alzheimer'sdisease.

31. The composition of any one of paragraphs 1 to 28 for neutralizing atoxin, virus, bacterium or allergen, with an antibody.

32. A method for treating or preventing a disease or disorderameliorated by a humoral immune response, said method comprising,consisting of, or consisting essentially of administering thecomposition of any one of paragraphs 1 to 28 to a subject.

33. A method for treating or preventing an infectious disease; a cancerinvolving a membrane surface-bound cancer antigen; or a disease ordisorder where it is desirable to sequester antigen in circulation, suchas Alzheimer's disease, said method comprising, consisting of, orconsisting essentially of administering the composition of any one ofparagraphs 1 to 28 to a subject.

34. The method of paragraph 33, wherein the infectious disease isinfluenza, a respiratory tract infection caused by human respiratorysyncytial virus, pertussis, anthrax or malaria.

35. A method for neutralizing a toxin, virus, bacterium or allergen,with an antibody, said method comprising, consisting of, or consistingessentially of administering the composition of any one of paragraphs 1to 28 to a subject.

36. The method of paragraph 35, wherein the toxin is a drug substance,such as cocaine.

37. The method of any one of paragraphs 32 to 36, wherein the subject isa mammal, preferably a human.

38. A kit useful for treating or preventing a disease or disorderameliorated by a humoral immune response; or useful for treating orpreventing an infectious disease; a cancer involving a membranesurface-bound cancer antigen; or a disease or disorder where it isdesirable to sequester antigen in circulation, such as Alzheimer'sdisease; or useful for neutralizing a toxin, virus, bacterium orallergen, with an antibody, wherein the kit comprises, consists of, orconsists essentially of the composition of any one of paragraphs 1 to28, and instructions for its use thereof.

39. A method of preparing the composition of paragraph 1, comprising,consisting of, or consisting essentially of: hydrating a homogenousmixture of S100 lecithin and cholesterol in the presence of the antigento form a liposome preparation with encapsulated antigen; extruding andthen mixing the liposome preparation with the adjuvant; lyophilizing andreconstituting the resultant product in a carrier comprising acontinuous phase of a hydrophobic substance. In an alternate embodiment,the hydrating step may be performed in the presence of a homogenousmixture of dioleoyl-phosphatidylcholine (DOPC) and cholesterol.

40. A method of preparing the composition of paragraph 1, comprising,consisting of, or consisting essentially of: hydrating a homogenousmixture of dioleoyl-phosphatidylcholine (DOPC) and cholesterol in thepresence of the antigen and the adjuvant to form a liposome preparationwith encapsulated antigen and adjuvant; lyophilizing and reconstitutingthe liposome preparation in a carrier comprising a continuous phase of ahydrophobic substance. In an alternate embodiment, the hydrating stepmay be performed in the presence of a homogenous mixture of S100lecithin and cholesterol.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1

Pathogen free, female CD1 mice, 6-8 weeks of age, were obtained fromCharles River Laboratories (St Constant, QC, Canada) and were housedaccording to institutional guidelines with water and food ad libitum,under filter controlled air circulation.

The H5N1 recombinant hemagglutinin protein, was purchased from ProteinSciences (Meridien, Conn., USA). This recombinant protein has anapproximate molecular weight of 72,000 daltons and corresponds to thehemagglutinin glycoprotein, an antigenic protein present on the surfaceof the H5N1 influenza virus. This recombinant protein, hereafterdesignated rHA, was used as a model antigen to test the efficacy ofvaccine formulations. rHA was used at 1 microgram per 50 microliterdose.

Vaccine efficacy was assessed by enzyme-linked immunosorbent assay(ELISA), a method that allows the detection of antigen-specific antibodylevels in the serum of immunized animals. Performing the ELISA on seracollected from immunized mice on a regular interval (every four weeksfor example), is useful for monitoring the antibody responses to a givenvaccine formulation. Briefly, a 96-well microtiter plate is coated withantigen (rHA, 1 microgram/milliliter) ovemight at 4 degrees Celsius,blocked with 3% gelatin for 30 minutes, then incubated overnight at 4degrees Celsius with serial dilutions of sera, typically starting at adilution of 1/2000. A secondary reagent (protein G conjugated toalkaline phosphatase, EMD chemicals, Gibbstown, N.J., USA) is then addedto each well at a 1/500 dilution for one hour at 37 degrees Celsius.Following a 60 minute incubation with a solution containing 1milligram/milliliter 4-nitrophenyl phosphate disodium salt hexahydrate(Sigma-Aldrich Chemie GmbH, Switzerland), the 405 nanometer absorbanceof each well is measured using a microtiter plate reader (ASYS HitechGmbH, Austria). Endpoint titers are calculated as described in Frey A.et al. (Journal of Immunological Methods, 1998, 221:35-41). Calculatedtiters represent the highest dilution at which a statisticallysignificant increase in absorbance is observed in serum samples fromimmunized mice versus serum samples from naïve, non-immunized controlmice. Titers are presented as log 10 values of the endpoint dilution.

To formulate vaccine corresponding to the invention, a 10:1 w:whomogenous mixture of S100 lecithin and cholesterol (Lipoid GmbH,Germany) was hydrated in the presence of rHA in phosphate buffer to formliposomes with encapsulated rHA. In brief, 20 micrograms of rHA in 775microliters of 50 millimolar phosphate buffer (pH 7.4) was added to 132milligrams of the S100 lecithin/cholesterol mixture to formapproximately 900 microliters of a liposome suspension encapsulating therHA antigen. The liposome preparation was then extruded by passing thematerial through a manual mini-extruder (Avanti, Alabaster, Ala., USA)fitted with a 200 nanometer polycarbonate membrane. To incorporate theadjuvant, the sized liposome mixture was thoroughly mixed with 20micrograms of Pam-3-Cys-Ser-(Lys)4 (SEQ ID NO: 1) (designated P3C)adjuvant (EMC Microcollections GmbH, Germany) in 100 microliters ofphosphate buffer. After diluting the final mixture in half using water,the liposome suspension was lyophilized using the Virtis Advantagefreeze dryer (SP Industries, Warminister, Pa., USA). For every 1milliliter of original liposome suspension containing rHA and P3C, 800microliters of a mineral oil carrier equivalent to Freund's incompleteadjuvant (known as Montanide® ISA 51, supplied by Seppic. France) wasused to reconstitute the lyophilized liposomes to form a water-freeliposome suspension. Each vaccine dose consisted of 50 microliters ofthe above described formulation containing liposomes, rHA antigen, P3Cadjuvant, and the mineral oil carrier. This vaccine formulation will bereferred to as water-free/liposome/P3C/hydrophobic carrier.

The efficacy of the water-free liposome formulation described above wascompared to the efficacy of a control vaccine consisting of 1 microgramof rHA and 50 micrograms of aluminum hydroxide (alhydrogel, Sigma,Mississauga, ON, Canada, hereafter named alum) in 50 microliters of 50millimolar phosphate buffer (pH 7.4). One group of mice (N=9) wereinjected once (no boosting) with 1 microgram of rHA antigen and 1microgram of P3C adjuvant formulated in 50 microliters of water-free/lliposome/P3C/hydrophobic carrier as described above. Group 2 mice (N=8)were vaccinated twice (day 0 and day 28) with 1 microgram of rHA and 50micrograms of alum adjuvant suspended in 50 millimolar phosphate buffer.All mice were vaccinated intramuscularly in the flank region and serumsamples were collected at 3, 4, and 8 weeks post-immunization. rHAantibody titers in these sera were examined by ELISA as described above.

The results of this experiment are shown in FIG. 1. Group 2 micegenerated a detectable antigen-specific antibody response following theadministration of an alum-adjuvanted control vaccine. Group 1 mice,vaccinated with the water-free/liposome/P3C/hydrophobic carrierformulation, yielded significantly enhanced endpoint titers compared tothose of group 2. Group 2 mice generated titers up to Ser. No.11/512,000 (log 10 value of 5.71) and up to 1/256,000 (log 10 value of5.41) at three and four weeks respectively (before boost) and up to1/4,096,000 (log 10 equal to 6.61) at eight weeks post-vaccination(after boost). The presence of such antibody responses confirms agenuine immune response generated as a result of the vaccination. Group1 mice, vaccinated with the vaccine corresponding to the invention, wereable to generate endpoint titers reaching up to 1/2,048,000 (log 10value of 6.31) at three and four weeks post-vaccination and 1/8,192,000(a log 10 value of 6.91) at eight weeks post-immunization. These resultsindicate that single dose water-free/l liposome/hydrophobic carrierformulations containing a palmitic acid adjuvant are capable ofgenerating an enhanced in vive immune response compared to a single(week 3 and week 4 data points) or boosted (week 8 data point), aqueousalum based control vaccine.

Example 2

Pathogen free, young adult female Balb/C mice were obtained from CharlesRiver Laboratories (St Constant, QC, Canada) and were housed accordingto institutional guidelines with water and food ad libitum, under filtercontrolled air circulation.

The pertussis toxoid protein was sourced from Biocine (ConnaughtBiosciences, Toronto, ON, Canada). This multi-subunit protein has anapproximate molecular weight of 106 Kilo-daltons and corresponds to anantigenic toxin produced by Bordetella pertussis, the causative bacteriaof whooping cough. This protein, hereafter designated PT, was used as amodel antigen to test the efficacy of vaccine formulations. PT was usedat 1 microgram per 50 microliter dose.

Vaccine efficacy was assessed by live bacterial challenge withBordetella pertussis. Mice were challenged by aerosol inoculation with9.1×10^8 Bordetella pertussis, 56 days post-vaccination. Several micewere sacrificed immediately to establish baseline bacterial lung counts.Remaining mice were monitored and sacrificed at eight and fifteen dayspost-challenge and bacterial lung counts established.

To formulate vaccine corresponding to the invention, a 10:1 w:whomogenous mixture of DOPC and cholesterol (Lipoid GmbH, Germany) washydrated in the presence of PT and Pam-3-Cys-Ser-(Lys)4 (SEQ ID NO:1)(designated P3C) in phosphate buffer to form liposomes withencapsulated PT and P3C. In brief, 20 micrograms each of PT and P3C in850 microliters of 50 millimolar phosphate buffer was added to 132milligrams of the S100 lecithin/cholesterol mixture to formapproximately one milliliter of a liposome suspension encapsulating thePT antigen and P3C adjuvant. The liposome preparation was thenlyophilized using the Virtis Advantage freeze dryer (SP Industries,Warminister, Pa., USA). For every one milliliter of original liposomesuspension containing rHA and P3C, 800 microliters of a mineral oilcarrier equivalent to Freund's incomplete adjuvant (known as Montanide®ISA 51, supplied by Seppic, France) was used to reconstitute thelyophilized liposomes to form a water-free liposome suspension. Eachvaccine dose consisted of 50 microliters of the above describedformulation containing liposomes, PT antigen, P3C adjuvant, and themineral oil carrier. This vaccine formulation will be referred to aswater-free/liposome/P3C/hydrophobic carrier.

The efficacy of the water-free liposome formulation described above wascompared to the efficacy of a control vaccine consisting of 1 microgramof PT and 100 micrograms of aluminum hydroxide adjuvant (Alhydrogel,Sigma, Mississauga, ON, Canada, hereafter named alum) in 100 microlitersof 50 millimolar phosphate buffer (pH 7.0). One group of mice (N=11)were injected once (no boosting) with 1 microgram of PT antigen and 1microgram of P3C adjuvant formulated in 50 microliters ofwater-free/liposome/P3C/hydrophobic carrier as described above. Group 2mice (N=9) and group 3 mice (N=9) were vaccinated once or three times(day 0, day 21, and day 31) with 1 microgram of PT and 100 micrograms ofalum adjuvant suspended in 100 microliters of phosphate buffer. Micewere vaccinated intramuscularly in the flank region. Group 4 mice (N=10)remained unvaccinated for the duration of the study. All mice werechallenged on day 56 post-immunization and bacterial lung countsestablished 8 and 15 days post-challenge as described above.

The results of this experiment are shown in FIG. 2. Group 4 (naïve) micewere not able to clear the infection, bacterial counts were as high as2.5×10≡cfu per lung at 8 days post-challenge and 4.7×10^3 cfu per lungat 15 days post-challenge. Group 2 mice, vaccinated with one dose of thealum-adjuvanted control vaccine, had bacterial lung counts as high as8.9×10^3 and 3.1×10^2 cfu per lung at 8 and 15 days post challengerespectively. Group 3 mice vaccinated with three doses of the controlhad lung counts as high as 3.5×10^3 and 1.8×10^3 cfu per lung at thesame respective time points. Group 1 mice, vaccinated with a single doseof the vaccine corresponding to the invention, had a bacterial lungcount as high as 6.2×10^4 cfu per lung at 8 days post-challenge and 0cfu per lung in all animals at day 15 post-challenge. A single dose ofthe vaccine corresponding to the invention effectively protected themice from bacterial challenge and allowed them to completely clear theinfection from the lungs.

Example 3

Pathogen free, female New Zealand White rabbits, 2-3 kg in weight, wereobtained from Charles River Laboratories (St Constant, QC, Canada) andwere housed according to institutional guidelines with water and food adlibitum, under filter controlled air circulation.

The anthrax recombinant Protective Antigen was purchased from ListBiologics (Campbell, Calif.). This recombinant protein has anapproximate molecular weight of 83,000 daltons and corresponds to theprotective antigen protein, a cell binding component of thethree-protein exotoxin produced by a Bacillus anthracis. Thisrecombinant protein, hereafter designated rPA, was used as a modelantigen to test the efficacy of vaccine formulations. rPA was used at 8micrograms per 100 microliter dose.

Vaccine efficacy was assessed by enzyme-linked immunosorbent assay(ELISA), a method that allows the detection of antigen-specific antibodylevels in the serum of immunized animals. Performing the ELISA on seracollected from immunized mice on a regular interval (every four weeksfor example), is useful for monitoring the antibody responses to a givenvaccine formulation. ELISA was performed as outlined in Example 1, usingrPA at 1 microgram/milliliter as the coating antigen.

To formulate vaccine corresponding to the invention, a 10:1 w:whomogenous mixture of DOPC lecithin and cholesterol (Lipoid GmbH,Germany) was hydrated in the presence of rPA and Pam-3-Cys (P3C) to formliposomes with encapsulated rPA and P3C. In brief, 80 micrograms of rPAand 20 micrograms of P3C in 850 microliters of sterile water were addedto 132 milligrams of the DOPC lecithin/cholesterol mixture to formapproximately one milliliter of a liposome suspension encapsulating therPA antigen and P3C adjuvant. After diluting to a sufficient quantityusing sterile water, the liposome suspension was lyophilized using theVirtis Advantage freeze dryer (SP Industries, Warminister, Pa., USA).For every 1 milliliter of original liposome suspension containing rPAand P3C, 800 microliters of a mineral oil carrier equivalent to Freund'sincomplete adjuvant (known as Montanide® ISA 51, supplied by Seppic,France) was used to reconstitute the lyophilized liposomes to form awater-free liposome suspension. Each vaccine dose consisted of 100microliters of the above described formulation containing liposomes, rPAantigen, P3C adjuvant, and the mineral oil carrier. This vaccine isdesignated Vaccine C (invention).

The efficacy of the vaccine formulation described above was compared tothe efficacy of a control vaccine consisting of 8 micrograms of rPA and350 micrograms of aluminum hydroxide (Alhydrogel) adjuvant (Sigma,Mississauga, ON, Canada) in 100 microliters of sterile water. One groupof rabbits (N=8) were injected once (no boosting) with 8 micrograms ofrPA antigen and 2 micrograms of P3C adjuvant formulated in 100microliters of vaccine formulation as described above (Group 1). Group 2rabbits (N=8) were vaccinated three times (day 0, 28 and 84) with 8microgram of rPA and 350 micrograms of alum adjuvant suspended insterile water. All rabbits were vaccinated intramuscularly in the rightgastrocnemius muscle and serum samples were collected at 3, 4, 8, 12 16,20 and 24 weeks post-immunization. rPA antibody titers in these serawere examined by ELISA as described above.

The results of this experiment are shown in FIG. 3. Group 2 rabbitsgenerated a detectable antigen-specific antibody response following theadministration of an alum-adjuvanted control vaccine. Group 1 rabbits,vaccinated with the Vaccine C formulation, yielded significantlyenhanced endpoint titers compared to those of group 2, at the early(pre-boost) time points. Group 2 rabbits generated titers up to 1/64,000(average log 10 value of 4.66) and up to 1/256,000 (average log 10 valueof 4.73) at three and four weeks respectively (before boost) and up to1/2,048,000 (average log 10 equal to 5.86) at eight weekspost-vaccination (after boost). The presence of such antibody responsesconfirms a genuine immune response generated as a result of thevaccination. Group 1 rabbits, vaccinated with the vaccine correspondingto the invention, were able to generate endpoint titers reaching up to1/2,048,000 (average log 10 value of 6.20 and 6.09) at three and fourweeks post-vaccination and 1/8,192,000 (average log 10 value of 6.53) ateight weeks post-immunization. These results showing that single doseliposome/hydrophobic carrier formulations containing a Pam-3-Cysadjuvant are capable of generating on average 34.6 times and 22.9 times(at three and four weeks respectively) more antibodies in vivo thancould be achieved with an aqueous alum control vaccine, demonstrate anability to produce a surprisingly strong immune response as early asthree weeks following a single vaccination.

Example 4

Pathogen free, female CD1 mice, 6-8 weeks of age, were obtained fromCharles River Laboratories (St Constant, QC, Canada) and were housedaccording to institutional guidelines with water and food ad libitum,under filter controlled air circulation.

The H5N1 recombinant hemagglutinin protein was purchased from ProteinSciences (Meridien, Conn., USA). This recombinant protein has anapproximate molecular weight of 72,000 daltons and corresponds to thehemagglutinin glycoprotein, an antigenic protein present on the surfaceof the H5N1 influenza virus. This recombinant protein, hereafterdesignated rHA, was used as a model antigen to test the efficacy ofvaccine formulations. rHA was used at 1 microgram per 50 microliterdose.

Vaccine efficacy was assessed by enzyme-linked immunosorbent assay(ELISA), a method that allows the detection of antigen-specific antibodylevels in the serum of immunized animals. Performing the ELISA on seracollected from immunized mice on a regular interval (every four weeksfor example), is useful for monitoring the antibody responses to a givenvaccine formulation. ELISA was carried out as described in Example 1.

To formulate vaccine corresponding to the invention, a 10:1 w:whomogenous mixture of S100 lecithin and cholesterol (Lipoid GmbH,Germany) was hydrated in the presence of rHA and Pam-3-Cys (P3C) inphosphate buffer to form liposomes with encapsulated rHA and P3C. Inbrief, 20 micrograms each of rHA and P3C in 850 microliters of 50millimolar phosphate buffer was added to 132 milligrams of the S100lecithin/cholesterol mixture to form approximately one milliliter of aliposome suspension encapsulating the rHA antigen and P3C adjuvant. Theliposome preparation was diluted to a sufficient quantity with sterilewater and then lyophilized using the Virtis Advantage freeze dryer (SPIndustries, Warminister, Pa., USA). For every one milliliter of originalliposome suspension containing rHA and P3C, 800 microliters of themineral oil carrier (Montanide® ISA 51, supplied by Seppic, France) wasused to reconstitute the lyophilized liposomes to form a water-freeliposome suspension. Each vaccine dose consisted of 50 microliters ofthe above described formulation containing liposomes, rHA antigen, P3Cadjuvant, and the mineral oil carrier. This vaccine formulation will bereferred to as liposome/P3C/hydrophobic carrier. This formulation wasused to vaccinate Group 1 mice (n=10).

Group 2 mice (n=10) were treated with 1 microgram of rHA and 1 microgramof P3C per 50 microliter dose, in the absence of liposomes/hydrophobiccarrier. Group 3 mice (n=10) were treated with 1 microgram rHA and 1microgram of P3C per 50 microliter dose formulated as anaqueous/liposome/P3C vaccine, in the absence of hydrophobic carrier.Group 4 mice (n=10) were treated with 1 microgram of rHA formulated as aliposome/hydrophobic carrier vaccine, in the absence of P3C. All micewere vaccinated intramuscularly in the flank region and serum sampleswere collected at 3, 4, 8, 12, 16 and 20 weeks post-immunization. rHAantibody titers in these sera were examined by ELISA as described.

The efficacies of these vaccine formulations were tested to evaluate therelative contribution of the components of these vaccine formulations(see FIG. 4). The titres from mice in all control groups (groups 2, 3and 4) were consistently lower than the titres from group 1, vaccinatedwith Vaccine A, indicating that all components of this formulation aremay be important for enhanced immunogenicity. For example, at week 8post-vaccination, mice in group 1 (vaccinated with Vaccine A) were ableto generate endpoint titers reaching up to 1/2,048,000 (average log 10value of 5.65), whereas mice in group 2, 3 and 4 were able to generateendpoint titers of 1/64,000 (average log 10 value of 4.41), 1/128,000(average log 10 value of 4.44) and 1/128,000 (average log 10 value of4.69), respectively. The titers generated by mice in group one weresignificantly higher (p<0.0001, by one way analysis of variance) thanthe titers generated in any of the three control vaccine groups. Thisindicates an involvement of all components of vaccine formulation A,specifically antigen, liposomes, a palmitic acid adjuvant and ahydrophobic carrier, in simulating maximal immunogenicity of thisformulation.

Example 5

Pathogen free, female Balb/C mice, 6-12 weeks of age, were obtained fromCharles River Laboratories (St Constant, QC, Canada) and were housedaccording to institutional guidelines with water and food ad libitum,under filter controlled air circulation.

The antigen used in vaccine formulations was a heat inactivatedInfluenza strain A/PR/8/34 (H1N1). A viral stock was prepared bypropagation in chicken eggs. An aliquot of A/PR/8/34 viral stock wasquickly thawed and placed in a 56 degree Celcius water bath for 30minutes to allow heat inactivation of the virus.

Vaccines were administered on day zero under isoflurane anesthetic,intramuscularly into the thigh muscle (one vaccine dose was divided intotwo injections, one per leg). The mice were weighed during the weekafter vaccination to ensure the vaccine itself did not cause illness.

On day 28, the mice were anesthetized using isoflurane and inoculatedintranasally with 10×MLD50 of virus (two separate administrations of 25microliters each divided equally into each nostril). The mice were thenmonitored for 10 days by measuring weight, temperature, and hydration,and by observing appearance, posture, and behavior. Mice that reachedpre-determined points of morbidity were euthanized.

Mice in group 1 (n=10) were vaccinated with saline only and served as anegative control vaccine.

Mice in group 2 (n=10) were vaccinated with 2.56×10^3 TCID50 of heatinactivated Influenza strain A/PR/8/34 (H1N1) formulated in Alhydrogel.

Mice in group 3 (n=10) were vaccinated with a liposome/P3C/hydrophobiccarrier vaccine. Briefly, a 10:1 (w:w) homogenous mixture of S100lecithin and cholesterol (Lipoid GmbH, Germany) was hydrated in thepresence of heat inactivated Influenza strain A/PR/8/34 and sterilewater to form approximately 850 microliters of liposomes withencapsulated antigen. Pam-3-Cys (P3C) adjuvant was then added, liposomesmixed well, and the mixture diluted to a sufficient quantity withsterile water before being lyophilized using the Virtis Advantage freezedryer (SP Industries, Warminister, Pa., USA). For every 1 milliliter oforiginal liposome suspension containing A/PR/8/34 and P3C, 800microliters of a mineral oil carrier (Montanide® ISA 51, Seppic. France)was used reconstitute the lyophilized liposomes to form a water-freeliposome suspension. Each dose volume was 50 microliters and containedliposomes, influenza strain A/PR/8/34 (2.56×10^3 TCID50), P3C (1microgram), and the mineral oil carrier.

The results of this experiment are shown in FIG. 5. Group 1 micevaccinated with saline alone rapidly developed clinical signs ofinfluenza infection and all succumbed to infection by day four. The micein group 2, vaccinated with antigen formulated in Alhydrogel,demonstrated moderately severe clinical symptoms upon influenzainfection, with 30% of animals succumbing to infection. However, themice in group 3, vaccinated with the liposome/P3C/hydrophobic carriervaccine, had relatively mild clinical symptoms and 100% survivedinfluenza infection.

These observations demonstrate that Pam-3-Cys formulated in the vaccineof the invention can enhance the immune response to inactivated viralvaccine formulations, as demonstrated by enhanced control of the virusupon infection.

Example 6

Pathogen free, female CD1 mice, 6-8 weeks of age, were obtained fromCharles River Laboratories (St Constant, QC, Canada) and were housedaccording to institutional guidelines with water and food ad libitum,under filter controlled air circulation.

As in Examples 1 and 4, H5N1 recombinant hemagglutinin protein,corresponding to the hemagglutinin glycoprotein on the surface of theH5N1 influenza virus, was purchased from Protein Sciences (Meridien,Conn., USA). This recombinant protein, hereafter designated rHA, wasused as a model antigen to test the efficacy of vaccine formulations.rHA was used at 1 microgram per 50 microliter dose.

Vaccine efficacy was assessed by enzyme-linked immunosorbent assay(ELISA), a method that allows the detection of antigen-specific antibodylevels in the serum of immunized animals. Performing the ELISA on seracollected from immunized mice on a regular interval (every four weeksfor example), is useful for monitoring the antibody responses to a givenvaccine formulation. ELISA was carried out as described in Example 1.

Both vaccines in this example were formulated as described in Example 4.In summary, for the vaccine corresponding to the invention, rHA antigenand Pam-3-Cys (P3C) adjuvant in 50 millimolar phosphate buffer were usedto hydrate S100 lecithin and cholesterol. The final liposome preparationwas lyophilized and then reconstituted with ISA51. The final vaccineconsisted of 50 microliters of a formulation containing liposomes, 1microgram of rHA antigen, 1 microgram P3C of adjuvant, and the mineraloil carrier. This vaccine formulation will be referred to asliposome/P3C/hydrophobic carrier. This formulation was used to vaccinateGroup 1 mice (n=9).

Group 2 mice (n=9) were treated with a formulation containing liposomes,1 microgram of rHA, 1 microgram of Imiquimod (IMQ) adjuvant, and themineral oil carrier. This vaccine will be referred to asliposome/IMQ/hydrophobic carrier. In summary, rHA antigen and IMQadjuvant (InvivoGen, San Diego, Calif., USA) in 50 millimolar phosphatebuffer were used to hydrate S100 lecithin and cholesterol. The finalliposome preparation was lyophilized and then reconstituted with ISA51.

All mice were vaccinated intramuscularly in the flank region and serumsamples were collected at 4 weeks post-immunization. rHA antibody titersin these sera were examined by ELISA as described in Example 1.

The efficacies of these vaccine formulations were tested to evaluate therelative contribution of the P3C adjuvant in the liposome/l hydrophobiccarrier formulation (see FIG. 6). Group 2 mice generated a detectableantigen-specific antibody response following the administration of anImiquimod-adjuvanted control vaccine. Group 1 mice, vaccinated with theliposome/P3C/hydrophobic carrier formulation, yielded significantlyenhanced endpoint titers compared to those of group 2 (P<0.005). Group 2mice generated titers up to 1/128,000 (log 10 value of 5.11) at 28 days(4 weeks) post-vaccination. As noted in example 1, the presence of suchantibody responses confirms a genuine immune response generated as aresult of the vaccination. Group 1 mice, vaccinated with the vaccinecorresponding to the invention, were able to generate endpoint titersreaching up to 1/1,024,000 (log 10 value of 6.01) at four weekspost-immunization. This data indicates that the vaccine corresponding tothe invention (liposomes/P3C/hydrophobic carrier) is able to stimulate aspecific humoral immune response which is significantly stronger than acomparable vaccine prepared with a different adjuvant(liposomes/IMQ/hydrophobic carrier).

Example 7

Pathogen free, female C57BL6 mice, 6-8 weeks of age, were obtained fromCharles River Laboratories (St Constant, QC, Canada) and were housedaccording to institutional guidelines with water and food ad libitum,under filter controlled air circulation.

Untreated “naïve” mice were terminated and spleens collected. A singlecell suspension was prepared from the splenocytes and red blood cellslysed using ACK lysis buffer. B cells were isolated using a negativeselection magnetic isolation kit from Miltenyi (Auburn, Calif., USA).Cells were resuspended in complete RPMI media containing 10% FBS, 1%penicillin-streptomycin, 1% L-glutamine and 0.1% b-mercaptoethanol(c-RPMI) at a final concentration of 2×10⁶ cells/mL. B cells were addedto wells of a 96-well plate (2×10⁵ cells/well) with anti-Ig (2.5 ug/mL;BD Biosciences, Mississauga, Canada) and anti-CD40 (1 ug/mL; BDBiosciences). B cells were stimulated with the following adjuvants intriplicate: Pam2Cys (EMC Microcollections, Tuebingen, Germany). Pam3Cys(EMC Microcollections), Poly I:C (Thermo Fisher, Mich., USA) and LPS(Sigma-Aldrich, Oakville, Canada), or no adjuvant. Each adjuvant wasdosed at 10 ug/mL, 1 ug/mL and 0.1 ug/mL, except LPS which was dosed at100 ng/mL, 10 ng/mL and 1 ng/mL. Cells were incubated at 37° C./5% CO₂for 3 days. Eighteen hours before the end of the experiment,[³H]-thymidine was added to each well at a final concentration of 0.2uCi/well. Plates were harvested using Titertek Cell Harvester (SkatronInstruments, Sterling, Va., USA) onto filter membranes which were thencounted with Beckman LS6000IC liquid scintillation counter (BeckmanCoulter Inc., Mississauga, ON, Canada). Proliferation was quantified byaveraging the triplicate counts per minute (CPM) representing theincorporation of [3H]-thymidine.

Results of adjuvant stimulation of B cells show that while Pam3Cys andPam2Cys can induce potent proliferation of B cells, Poly I:C does not(see FIG. 7). LPS, included as a positive control, is known to induce Bcell proliferation of low concentrations. Based on these results, it isreasonable to expect that vaccines containing Pam2Cys or Pam3Cys wouldhave similar effects on B cells in vivo, and would be capable offacilitating the production of similar levels of antigen specificantibodies.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

It must be noted that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise. Unless defined otherwiseall technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to encompass the same meaning as “and/or” as defined above.For example, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items.

As used herein, whether in the specification or the appended claims, thetransitional terms “comprising”, “including”, “carrying”, “having”,“containing”, “involving”, and the like are to be understood as beinginclusive or open-ended (i.e., to mean including but not limited to),and they do not exclude unrecited elements, materials or method steps.Only the transitional phrases “consisting of” and “consistingessentially of”, respectively, are closed or semi-closed transitionalphrases with respect to claims. The transitional phrase “consisting of”excludes any element, step, or ingredient which is not specificallyrecited. The transitional phrase “consisting essentially of” limits thescope to the specified elements, materials or steps and to those that donot materially affect the basic characteristic(s) of the inventiondisclosed and/or claimed herein.

The invention claimed is:
 1. A method for vaccinating against aninfectious disease by inducing a protective humoral immune responseagainst a pathogen of the infectious disease, said method comprisingadministering to a subject an effective amount of a water-freecomposition comprising an adjuvant that activates or increases theactivity of toll-like receptor 2 (TLR2) and dehydrated liposomes with anencapsulated antigen of the pathogen, reconstituted in a mineral oilcarrier, wherein the antigen comprises a B cell epitope and is capableof inducing a humoral immune response; wherein the adjuvant comprisesPAM₂Cys-Ser-(Lys)4 (SEQ ID NO: 1) or PAM₃Cys-Ser-(Lys)4 (SEQ ID NO:1);and wherein the antigen is not coupled covalently or non-covalently tothe adjuvant.
 2. The method of claim 1, wherein the adjuvant activatesor increases the activity of TLR2 by interacting with a TLR2 dimer ofTLR1/2 or TLR2/6.
 3. The method of claim 1, wherein the adjuvant isPAM₂Cys-Ser-(Lys)4.
 4. The method of claim 1, wherein the adjuvant isPAM₃Cys-Ser-(Lys)
 4. 5. The method of claim 1, wherein the antigen is apolypeptide or a carbohydrate.
 6. The method of claim 1, wherein the Bcell epitope is derived from a virus or bacteria.
 7. The method of claim6, wherein the B cell epitope is derived from influenza virus,Bordetella pertussi, or Bacillus anthracis.
 8. The method of claim 7,wherein the B cell epitope is an epitope of a hemagglutinin protein ofH5N1 influenza virus, an epitope of pertussis toxoid protein, or anepitope of an anthrax recombinant protective antigen.
 9. The method ofclaim 1, wherein both the antigen and the adjuvant are encapsulated inthe dehydrated liposomes reconstituted in the mineral oil carrier. 10.The method of claim 1, wherein the liposomes are non-cationic liposomes,wherein the lipids of the liposomes consist of neutral lipids,negatively charged lipids or a mixture thereof.
 11. The method of claim1, wherein the composition is capable of inducing a humoral immuneresponse with a single dose.
 12. The method of claim 1, wherein theinfectious disease is selected from the group consisting of influenza, arespiratory tract infection or lung infection caused by respiratorysyncytial virus, whooping cough, anthrax, and malaria.
 13. The method ofclaim 1, wherein the liposomes are comprised of phospholipids andcholesterol.
 14. The method of claim 1, wherein the carrier is a mannideoleate in mineral oil solution.
 15. The method of claim 14, wherein thecarrier is Montanide® ISA
 51. 16. The method of claim 1, wherein theantigen is selected from the group consisting of H5N1 recombinanthemagglutinin protein, pertussis toxoid protein, anthrax recombinantProtective Antigen and heat inactivated Influenza strain A/PR/8/34(H1N1).
 17. The method of claim 1, wherein the liposomes comprise S100lecithin and cholesterol or dioleoyl-phosphatidylcholine (DOPC) andcholesterol; the carrier is Montanide® ISA 51; and the adjuvant isPAM₃Cys-Ser-(Lys) 4 (SEQ ID NO:1).
 18. The method of claim 17, whereinthe antigen is selected from the group consisting of H5N1 recombinanthemagglutinin protein, pertussis toxoid protein, anthrax recombinantProtective Antigen and heat inactivated Influenza strain A/PR/8/34(H1N1).
 19. The method of claim 1, wherein the liposomes are dehydratedby lyophilization.
 20. The method of claim 9, wherein the liposomes aredehydrated by lyophilization.